Ligand

ABSTRACT

The invention provides a dual-specific ligand comprising a first immunoglobulin variable domain having a first binding specificity and a complementary or non-complementary immunoglobulin variable domain having a second binding specificity.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/704,832, filed Feb. 8, 2007, which is a continuation in part of U.S.application Ser. No. 11/023,959, filed Dec. 28, 2004, which is acontinuation of International Application PCT/GB2003/002804, filed 30Jun. 2003, which claims the priority of PCT/GB02/03014, filed 28 Jun.2002 and Great Britain Application GB 0230202.4, filed 27 Dec. 2002, thecontents of which are incorporated herein by reference. This applicationis also a continuation in part of WO2005118642, filed May 31, 2005,which claims the benefit of U.S. 60/576,271 filed Jun. 1, 2004, and U.S.60/632,361 filed Dec. 2, 2004, the contents of which are incorporatedherein by reference. The application also claims priority from U.S. Ser.No. 11/704,832, filed 8 Feb. 2007.

The present invention relates to dual specific ligands. In particular,the invention provides a method for the preparation of dual-specificligands comprising a first immunoglobulin single variable domain bindingto a first antigen or epitope, and a second immunoglobulin singlevariable domain binding to a second antigen or epitope. Moreparticularly, the invention relates to dual-specific ligands whereinbinding to at least one of the first and second antigens or epitopesacts to increase the half-life of the ligand in vivo. Open and closedconformation ligands comprising more than one binding specificity aredescribed.

INTRODUCTION

The antigen binding domain of an antibody comprises two separateregions: a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L): which can be either V_(κ) or V_(λ)). The antigenbinding site itself is formed by six polypeptide loops: three from V_(H)domain (H1, H2 and H3) and three from V_(L) domain (L1, L2 and L3). Adiverse primary repertoire of V genes that encode the V_(H) and V_(L)domains is produced by the combinatorial rearrangement of gene segments.The V_(H) gene is produced by the recombination of three gene segments,V_(H), D and J_(H). In humans, there are approximately 51 functionalV_(H) segments (Cook and Tomlinson (1995) Immunol Today, 16: 237), 25functional D segments (Corbett et al. (1997) J. Mol. Biol., 268: 69) and6 functional J_(H) segments (Ravetch et al. (1981) Cell, 27: 583),depending on the haplotype. The V_(H) segment encodes the region of thepolypeptide chain which forms the first and second antigen binding loopsof the V_(H) domain (H1 and H2), whilst the V_(H), D and J_(H) segmentscombine to form the third antigen binding loop of the V_(H) domain (H3).The V_(L) gene is produced by the recombination of only two genesegments, V_(L) and J_(L). In humans, there are approximately 40functional V_(κ) segments (Schable and Zachau (1993) Biol. Chem.Hoppe-Seyler, 374: 1001), 31 functional V_(λ) segments (Williams et al.(1996) J. Mol. Biol., 264: 220; Kawasaki et al. (1997) Genome Res., 7:250), 5 functional J_(κ) segments (Hieter et al. (1982) J. Biol. Chem.,257: 1516) and 4 functional J_(λ) segments (Vasicek and Leder (1990) J.Exp. Med., 172: 609), depending on the haplotype. The V_(L) segmentencodes the region of the polypeptide chain which forms the first andsecond antigen binding loops of the V_(L) domain (L1 and L2), whilst theV_(L) and J_(L) segments combine to form the third antigen binding loopof the V_(L) domain (L3). Antibodies selected from this primaryrepertoire are believed to be sufficiently diverse to bind almost allantigens with at least moderate affinity. High affinity antibodies areproduced by “affinity maturation” of the rearranged genes, in whichpoint mutations are generated and selected by the immune system on thebasis of improved binding.

Analysis of the structures and sequences of antibodies has shown thatfive of the six antigen binding loops (H1, H2, L1, L2, L3) possess alimited number of main-chain conformations or canonical structures(Chothia and Lesk (1987) J. Mol. Biol., 196: 901; Chothia et al. (1989)Nature, 342: 877). The main-chain conformations are determined by (i)the length of the antigen binding loop, and (ii) particular residues, ortypes of residue, at certain key position in the antigen binding loopand the antibody framework. Analysis of the loop lengths and keyresidues has enabled us to the predict the main-chain conformations ofH1, H2, L1, L2 and L3 encoded by the majority of human antibodysequences (Chothia et al. (1992) J. Mol. Biol., 227: 799; Tomlinson etal. (1995) EMBO 1, 14: 4628; Williams et al. (1996) J. Mol. Biol., 264:220). Although the H3 region is much more diverse in terms of sequence,length and structure (due to the use of D segments), it also forms alimited number of main-chain conformations for short loop lengths whichdepend on the length and the presence of particular residues, or typesof residue, at key positions in the loop and the antibody framework(Martin et al. (1996) J. Mol. Biol., 263: 800; Shirai et al. (1996) FEBSLetters, 399: 1.

Bispecific antibodies comprising complementary pairs of V_(H) and V_(L)regions are known in the art. These bispecific antibodies must comprisetwo pairs of V_(H) and V_(L)s, each V_(H)/V_(L) pair binding to a singleantigen or epitope. Methods described involve hybrid hybridomas(Milstein & Cuello A C, Nature 305:537-40), minibodies (Hu et al.,(1996) Cancer Res 56:3055-3061), diabodies (Holliger et al., (1993)Proc. Natl. Acad. Sci. USA 90, 6444-6448; WO 94/13804), chelatingrecombinant antibodies (CRAbs; (Neri et al., (1995) J. Mol. Biol. 246,367-373), biscFv (e.g. Atwell et al., (1996) Mol. Immunol. 33,1301-1312), “knobs in holes” stabilised antibodies (Carter et al.,(1997) Protein Sci. 6, 781-788). In each case each antibody speciescomprises two antigen-binding sites, each fashioned by a complementarypair of V_(H) and V_(L) domains. Each antibody is thereby able to bindto two different antigens or epitopes at the same time, with the bindingto EACH antigen or epitope mediated by a V_(H) and its complementaryV_(L) domain. Each of these techniques presents its particulardisadvantages; for instance in the case of hybrid hybridomas, inactiveV_(H)/V_(L) pairs can greatly reduce the fraction of bispecific IgG.Furthermore, most bispecific approaches rely on the association of thedifferent V_(H)/V_(L) pairs or the association of V_(H) and V_(L) chainsto recreate the two different binding sites. It is therefore impossibleto control the ratio of binding sites to each antigen or epitope in theassembled molecule and thus many of the assembled molecules will bind toone antigen or epitope but not the other. In some cases it has beenpossible to engineer the heavy or light chains at the sub-unitinterfaces (Carter et al., 1997) in order to improve the number ofmolecules which have binding sites to both antigens or epitopes but thisnever results in all molecules having binding to both antigens orepitopes.

There is some evidence that two different antibody binding specificitiesmight be incorporated into the same binding site, but these generallyrepresent two or more specificities that correspond to structurallyrelated antigens or epitopes or to antibodies that are broadlycross-reactive. For example, cross-reactive antibodies have beendescribed, usually where the two antigens are related in sequence andstructure, such as hen egg white lysozyme and turkey lysozyme(McCafferty et al., WO 92/01047) or to free hapten and to haptenconjugated to carrier (Griffiths A D et al. EMBO J 1994 13:14 3245-60).In a further example, WO 02/02773 (Abbott Laboratories) describesantibody molecules with “dual specificity”. The antibody moleculesreferred to are antibodies raised or selected against multiple antigens,such that their specificity spans more than a specifies a single bindingspecificity for two or more structurally related antigens; the V_(H) andV_(L) domains in such complementary pairs do not each possess a separatespecificity. The antibodies thus have a broad single specificity whichencompasses two antigens, which are structurally related. Furthermorenatural autoantibodies have been described that are polyreactive (Casali& Notkins, Ann. Rev. Immunol. 7, 515-531), reacting with at least two(usually more) different antigens or epitopes that are not structurallyrelated. It has also been shown that selections of random peptiderepertoires using phage display technology on a monoclonal antibody willidentify a range of peptide sequences that fit the antigen binding site.Some of the sequences are highly related, fitting a consensus sequence,whereas others are very different and have been termed mimotopes (Lane &Stephen, Current Opinion in Immunology, 1993, 5, 268-271). It istherefore clear that a natural four-chain antibody, comprisingassociated and complementary V_(H) and V_(L) domains, has the potentialto bind to many different antigens from a large universe of knownantigens. It is less clear how to create a binding site to two givenantigens in the same antibody, particularly those which are notnecessarily structurally related.

Protein engineering methods have been suggested that may have a bearingon this. For example it has also been proposed that a catalytic antibodycould be created with a binding activity to a metal ion through onevariable domain, and to a hapten (substrate) through contacts with themetal ion and a complementary variable domain (Barbas et al., 1993 Proc.Natl. Acad. Sci USA 90, 6385-6389). However in this case, the bindingand catalysis of the substrate (first antigen) is proposed to requirethe binding of the metal ion (second antigen). Thus the binding to theV_(H)/V_(L) pairing relates to a single but multi-antigen.

Methods have been described for the creation of bispecific antibodiesfrom camel antibody heavy chain single domains in which binding contactsfor one antigen are created in one variable domain, and for a secondantigen in a second variable domain. However, the variable domains werenot complementary. Thus a first heavy chain variable domain is selectedagainst a first antigen, and a second heavy chain variable domainagainst a second antigen, and then both domains are linked together onthe same chain to give a bispecific antibody fragment (Conrath et al.,J. Biol. Chem. 270, 27589-27594). However, the camel heavy chain singledomains are unusual in that they are derived from natural camelantibodies which have no light chains, and indeed the heavy chain singledomains are unable to associate with camel light chains to formcomplementary V_(H) and V_(L) pairs.

Single heavy chain variable domains have also been described, derivedfrom natural antibodies which are normally associated with light chains(from monoclonal antibodies or from repertoires of domains; seeEP-A-0368684). These heavy chain variable domains have been shown tointeract specifically with one or more related antigens, but have notbeen combined with other heavy or light chain variable domains to createa ligand with a specificity for two or more different antigens.Furthermore, these single domains have been shown to have a very shortin vivo half-life. Therefore such domains are of limited therapeuticvalue.

It has been suggested to make bispecific antibody fragments by linkingheavy chain variable domains of different specificity together (asdescribed above). The disadvantage with this approach is that isolatedantibody variable domains may have a hydrophobic interface that normallymakes interactions with the light chain and is exposed to solvent andmay be “sticky” allowing the single domain to bind to hydrophobicsurfaces. Furthermore, in the absence of a partner light chain thecombination of two or more different heavy chain variable domains andtheir association, possibly via their hydrophobic interfaces, mayprevent them from binding to one if not both of the ligands they areable to bind in isolation. Moreover, in this case the heavy chainvariable domains would not be associated with complementary light chainvariable domains and thus may be less stable and readily unfold (Worn &Pluckthun, 1998 Biochemistry 37, 13120-7).

SUMMARY OF THE INVENTION

The inventors have described, in their copending international patentapplication WO 03/002609 as well as copending unpublished UK patentapplication 0230203.2, dual specific immunoglobulin ligands whichcomprise immunoglobulin single variable domains which each havedifferent specificities. The domains may act in competition with eachother or independently to bind antigens or epitopes on target molecules.

In a first configuration, the present invention provides a furtherimprovement in dual specific ligands as developed by the presentinventors, in which one specificity of the ligand is directed towards aprotein or polypeptide present in vivo in an organism which can act toincrease the half-life of the ligand by binding to it.

Accordingly, in a first aspect, there is provided a dual-specific ligandcomprising a first immunoglobulin single variable domain having abinding specificity to a first antigen or epitope and a secondcomplementary immunoglobulin single variable domain having a bindingactivity to a second antigen or epitope, wherein one or both of saidantigens or epitopes acts to increase the half-life of the ligand invivo and wherein said first and second domains lack mutuallycomplementary domains which share the same specificity, provided thatsaid dual specific ligand does not consist of an anti-HSA V_(H) domainand an anti-β galactosidase V_(κ) domain. Preferably, neither of thefirst or second variable domains binds to human serum albumin (HSA).

Antigens or epitopes which increase the half-life of a ligand asdescribed herein are advantageously present on proteins or polypeptidesfound in an organism in vivo. Examples include extracellular matrixproteins, blood proteins, and proteins present in various tissues in theorganism. The proteins act to reduce the rate of ligand clearance fromthe blood, for example by acting as bulking agents, or by anchoring theligand to a desired site of action. Examples of antigens/epitopes whichincrease half-life in vivo are given in Annex 1 below.

Increased half-life is useful in in vivo applications ofimmunoglobulins, especially antibodies and most especially antibodyfragments of small size. Such fragments (Fvs, disulphide bonded Fvs,Fabs, scFvs, dAbs) suffer from rapid clearance from the body; thus,whilst they are able to reach most parts of the body rapidly, and arequick to produce and easier to handle, their in vivo applications havebeen limited by their only brief persistence in vivo. The inventionsolves this problem by providing increased half-life of the ligands invivo and consequently longer persistence times in the body of thefunctional activity of the ligand.

Methods for pharmacokinetic analysis and determination of ligandhalf-life will be familiar to those skilled in the art. Details may befound in Kenneth, A et al: Chemical Stability of Pharmaceuticals: AHandbook for Pharmacists and in Peters et al, Pharmacokinetc analysis: APractical Approach (1996). Reference is also made to “Pharmacokinetics”,M Gibaldi & D Perron, published by Marcel Dekker, 2^(nd) Rev. ex edition(1982), which describes pharmacokinetic parameters such as t alpha and tbeta half lives and area under the curve (AUC).

Half lives (t½ alpha and t½ beta) and AUC can be determined from a curveof serum concentration of ligand against time. The WinNonlin analysispackage (available from Pharsight Corp., Mountain View, Calif. 94040,USA) can be used, for example, to model the curve. In a first phase (thealpha phase) the ligand is undergoing mainly distribution in thepatient, with some elimination. A second phase (beta phase) is theterminal phase when the ligand has been distributed and the serumconcentration is decreasing as the ligand is cleared from the patient.The t alpha half life is the half life of the first phase and the t betahalf life is the half life of the second phase. Thus, advantageously,the present invention provides a ligand or a composition comprising aligand according to the invention having a tα half-life in the range of15 minutes or more. In one embodiment, the lower end of the range is 30minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, oralternatively, a ligand or composition according to the invention willhave a ta half life in the range of up to and including 12 hours. In oneembodiment, the upper end of the range is 11, 10, 9, 8, 7, 6 or 5 hours.An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4hours.

Advantageously, the present invention provides a ligand or a compositioncomprising a ligand according to the invention having a tβ half-life inthe range of 2.5 hours or more. In one embodiment, the lower end of therange is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11hours, or 12 hours. In addition, or alternatively, a ligand orcomposition according to the invention has a tβ half-life in the rangeof up to and including 21 days. In one embodiment, the upper end of therange is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or20 days. Advantageously a ligand or composition according to theinvention will have a tβ half life in the range 12 to 60 hours. In afurther embodiment, it will be in the range 12 to 48 hours. In a furtherembodiment still, it will be in the range 12 to 26 hours.

In addition, or alternatively to the above criteria, the presentinvention provides a ligand or a composition comprising a ligandaccording to the invention having an AUC value (area under the curve) inthe range of 1 mg/min/ml or more. In one embodiment, the lower end ofthe range is 5, 10, 15, 20, 30, 100, 200 or 300 mg/min/ml. In addition,or alternatively, a ligand or composition according to the invention hasan AUC in the range of up to 600 mg/min/ml. In one embodiment, the upperend of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg/min/ml.Advantageously, a ligand according to the invention will have a AUC inthe range selected from, but preferably not limited to, the groupconsisting of the following: 15 to 150 mg/min/ml, 15 to 100 mg/min/ml,15 to 75 mg/min/ml, and 15 to 50 mg/min/ml.

In a first embodiment, the dual specific ligand comprises twocomplementary variable domains, i.e. two variable domains that, in theirnatural environment, are capable of operating together as a cognate pairor group, even if in the context of the present invention they bindseparately to their cognate epitopes. For example, the complementaryvariable domains may be immunoglobulin heavy chain and light chainvariable domains (V_(H) and V_(L)). V_(H) and V_(L) domains areadvantageously provided by scFv or Fab antibody fragments. Variabledomains may be linked together to form multivalent ligands by, forexample: provision of a hinge region at the C-terminus of each V domainand disulphide bonding between cysteines in the hinge regions; orprovision of dAbs each with a cysteine at the C-terminus of the domain,the cysteines being disulphide bonded together; or production of V-CH &V-CL to produce a Fab format; or use of peptide linkers (for example,Gly₄Ser linkers discussed herein below) to produce dimers, trimers andfurther multimers.

The inventors have found that the use of complementary variable domainsallows the two domain surfaces to pack together and be sequestered fromthe solvent. Furthermore, the complementary domains are able tostabilise each other. In addition, it allows the creation ofdual-specific IgG antibodies without the disadvantages of hybridhybridomas as used in the prior art, or the need to engineer heavy orlight chains at the sub-unit interfaces. The dual-specific ligands ofthe first aspect of the present invention have at least one V_(H)/V_(L)pair. A bispecific IgG according to this invention will thereforecomprise two such pairs, one pair on each arm of the Y-shaped molecule.Unlike conventional bispecific antibodies or diabodies, therefore, wherethe ratio of chains used is determinative in the success of thepreparation thereof and leads to practical difficulties, the dualspecific ligands of the invention are free from issues of chain balance.Chain imbalance in conventional bi-specific antibodies results from theassociation of two different V_(L) chains with two different V_(H)chains, where V_(L) chain 1 together with V_(H) chain 1 is able to bindto antigen or epitope 1 and V_(L) chain 2 together with V_(H) chain 2 isable to bind to antigen or epitope 2 and the two correct pairings are insome way linked to one another. Thus, only when V_(L) chain 1 is pairedwith V_(H) chain 1 and V_(L) chain 2 is paired with V_(H) chain 2 in asingle molecule is bi-specificity created. Such bi-specific moleculescan be created in two different ways. Firstly, they can be created byassociation of two existing V_(H)/V_(L) pairings that each bind to adifferent antigen or epitope (for example, in a bi-specific IgG). Inthis case the V_(H)/V_(L) pairings must come all together in a 1:1 ratioin order to create a population of molecules all of which arebi-specific. This never occurs (even when complementary CH domain isenhanced by “knobs into holes” engineering) leading to a mixture ofbi-specific molecules and molecules that are only able to bind to oneantigen or epitope but not the other. The second way of creating abi-specific antibody is by the simultaneous association of two differentV_(H) chain with two different V_(L) chains (for example in abi-specific diabody). In this case, although there tends to be apreference for V_(L) chain 1 to pair with V_(H) chain 1 and V_(L) chain2 to pair with V_(H) chain 2 (which can be enhanced by “knobs intoholes” engineering of the V_(L) and V_(H) domains), this paring is neverachieved in all molecules, leading to a mixed formulation wherebyincorrect pairings occur that are unable to bind to either antigen orepitope.

Bi-specific antibodies constructed according to the dual-specific ligandapproach according to the first aspect of the present invention overcomeall of these problems because the binding to antigen or epitope 1resides within the V_(H) or V_(L) domain and the binding to antigen orepitope 2 resides with the complementary V_(L) or V_(H) domain,respectively. Since V_(H) and V_(L) domains pair on a 1:1 basis allV_(H)/V_(L) pairings will be bi-specific and thus all formatsconstructed using these V_(H)/V_(L) pairings (Fv, scFvs, Fabs,minibodies, IgGs, etc.) will have 100% bi-specific activity.

In the context of the present invention, first and second “epitopes” areunderstood to be epitopes which are not the same and are not bound by asingle monospecific ligand. In the first configuration of the invention,they are advantageously on different antigens, one of which acts toincrease the half-life of the ligand in vivo. Likewise, the first andsecond antigens are advantageously not the same.

The dual specific ligands of the invention do not include ligands asdescribed in WO 02/02773. Thus, the ligands of the present invention donot comprise complementary V_(H)/V_(L) pairs which bind any one or moreantigens or epitopes co-operatively. Instead, the ligands according tothe first aspect of the invention comprise a V_(H)/V_(L) complementarypair, wherein the V domains have different specificities.

Moreover, the ligands according to the first aspect of the inventioncomprise V_(H)/V_(L) complementary pairs having different specificitiesfor non-structurally related epitopes or antigens. Structurally relatedepitopes or antigens are epitopes or antigens which possess sufficientstructural similarity to be bound by a conventional V_(H)/V_(L)complementary pair which acts in a co-operative manner to bind anantigen or epitope; in the case of structurally related epitopes, theepitopes are sufficiently similar in structure that they “fit” into thesame binding pocket formed at the antigen binding site of theV_(H)/V_(L) dimer.

In a second aspect, the present invention provides a ligand comprising afirst immunoglobulin variable domain having a first antigen or epitopebinding specificity and a second immunoglobulin variable domain having asecond antigen or epitope binding specificity, wherein one or both ofsaid first and second variable domains bind to an antigen whichincreases the half-life of the ligand in vivo, and the variable domainsare not complementary to one another.

In one embodiment, binding to one variable domain modulates the bindingof the ligand to the second variable domain.

In this embodiment, the variable domains may be, for example, pairs ofV_(H) domains or pairs of V_(L) domains. Binding of antigen at the firstsite may modulate, such as enhance or inhibit, binding of an antigen atthe second site. For example, binding at the first site at leastpartially inhibits binding of an antigen at a second site. In such anembodiment, the ligand may for example be maintained in the body of asubject organism in vivo through binding to a protein which increasesthe half-life of the ligand until such a time as it becomes bound to thesecond target antigen and dissociates from the half-life increasingprotein.

Modulation of binding in the above context is achieved as a consequenceof the structural proximity of the antigen binding sites relative to oneanother. Such structural proximity can be achieved by the nature of thestructural components linking the two or more antigen binding sites,e.g., by the provision of a ligand with a relatively rigid structurethat holds the antigen binding sites in close proximity. Advantageously,the two or more antigen binding sites are in physically close proximityto one another such that one site modulates the binding of antigen atanother site by a process which involves steric hindrance and/orconformational changes within the immunoglobulin molecule.

The first and the second antigen binding domains may be associatedeither covalently or non-covalently. In the case that the domains arecovalently associated, then the association may be mediated for exampleby disulphide bonds or by a polypeptide linker such as (Gly₄Ser)_(n),where n=from 1 to 8, e.g., 2, 3, 4, 5 or 7.

Ligands according to the invention may be combined intonon-immunoglobulin multi-ligand structures to form multivalentcomplexes, which bind target molecules with the same antigen, therebyproviding superior avidity, while at least one variable domain binds anantigen to increase the half life of the multimer. For example, naturalbacterial receptors such as SpA have been used as scaffolds for thegrafting of CDRs to generate ligands which bind specifically to one ormore epitopes. Details of this procedure are described in U.S. Pat. No.5,831,012. Other suitable scaffolds include those based on fibronectinand Affibodies™. Details of suitable procedures are described in WO98/58965. Other suitable scaffolds include lipocallin and CTLA4, asdescribed in van den Beuken et al., J. Mol. Biol. (2001) 310, 591-601,and scaffolds such as those described in WO0069907 (Medical ResearchCouncil), which are based for example on the ring structure of bacterialGroEL or other chaperone polypeptides.

Protein scaffolds may be combined; for example, CDRs may be grafted onto a CTLA4 scaffold and used together with immunoglobulin V_(H) or V_(L)domains to form a ligand. Likewise, fibronectin, lipocallin and otherscaffolds may be combined.

In the case that the variable domains are selected from V-generepertoires selected for instance using phage display technology asherein described, then these variable domains can comprise a universalframework region, such that is they may be recognised by a specificgeneric ligand as herein defined. The use of universal frameworks,generic ligands and the like is described in WO99/20749. In the presentinvention, reference to phage display includes the use of both phageand/or phagemids.

Where V-gene repertoires are used, variation in polypeptide sequence ispreferably located within the structural loops of the variable domains.The polypeptide sequences of either variable domain may be altered byDNA shuffling or by mutation in order to enhance the interaction of eachvariable domain with its complementary pair.

In a preferred embodiment of the invention the ‘dual-specific ligand’ isa single chain Fv fragment. In an alternative embodiment of theinvention, the ‘dual-specific ligand’ consists of a Fab region of anantibody. The term “Fab region” includes a Fab-like region where two VHor two VL domains are used.

The variable domains may be derived from antibodies directed againsttarget antigens or epitopes. Alternatively they may be derived from arepertoire of single antibody domains such as those expressed on thesurface of filamentous bacteriophage. Selection may be performed asdescribed below.

In a third aspect, the invention provides a method for producing aligand comprising a first immunoglobulin single variable domain having afirst binding specificity and a second single immunoglobulin singlevariable domain having a second (different) binding specificity, one orboth of the binding specificities being specific for an antigen whichincreases the half-life of the ligand in vivo, the method comprising thesteps of:

(a) selecting a first variable domain by its ability to bind to a firstepitope,(b) selecting a second variable domain by its ability to bind to asecond epitope,(c) combining the variable domains; and(d) selecting the ligand by its ability to bind to said first epitopeand to said second epitope.

The ligand can bind to the first and second epitopes eithersimultaneously or, where there is competition between the bindingdomains for epitope binding, the binding of one domain may preclude thebinding of another domain to its cognate epitope. In one embodiment,therefore, step (d) above requires simultaneous binding to both firstand second (and possibly further) epitopes; in another embodiment, thebinding to the first and second epitopes is not simultaneous.

The epitopes are preferably on separate antigens.

Ligands advantageously comprise V_(H)/V_(L) combinations, or V_(H)/V_(H)or V_(L)/V_(L) combinations of immunoglobulin variable domains, asdescribed above. The ligands may moreover comprise camelid V_(HH)domains, provided that the V_(HH) domain which is specific for anantigen which increases the half-life of the ligand in vivo does notbind Hen egg white lysozyme (HEL), porcine pancreatic alpha-amylase orNmC-A; hcg, BSA-linked RR6 azo dye or S. mutans HG982 cells, asdescribed in Conrath et al., (2001) JBC 276:7346-7350 and WO99/23221,neither of which describe the use of a specificity for an antigen whichincreases half-life to increase the half life of the ligand in vivo.

In one embodiment, said first variable domain is selected for binding tosaid first epitope in absence of a complementary variable domain. In afurther embodiment, said first variable domain is selected for bindingto said first epitope/antigen in the presence of a third variable domainin which said third variable domain is different from said secondvariable domain and is complementary to the first domain. Similarly, thesecond domain may be selected in the absence or presence of acomplementary variable domain.

Antigens or epitopes targeted by the ligands of the invention whichincrease the half-life of a ligand, are not limited to serum albumintargets. Other embodiments of antigens or epitopes targeted by theligands of the invention which increase the half-life of a ligand invivo include, but are preferably not limited to, those antigens andepitopes listed in Annex 1 below

The antigens or epitopes targeted by the ligands of the invention, inaddition to the half-life enhancing protein, may be any antigen orepitope, but advantageously is an antigen or epitope that is targetedwith therapeutic benefit. The invention provides ligands, including openconformation, closed conformation and isolated dAb monomer ligands,specific for any such target, particularly those targets furtheridentified herein. Such targets may be, or be part of, polypeptides,proteins or nucleic acids, which may be naturally occurring orsynthetic. In this respect, the ligand of the invention may bind theepitope or antigen and act as an antagonist or agonist (e.g., EPOreceptor agonist). One skilled in the art will appreciate that thechoice is large and varied. They may be for instance, human or animalproteins, cytokines, cytokine receptors, where cytokine receptorsinclude receptors for cytokines, enzymes, co-factors for enzymes or DNAbinding proteins. Suitable cytokines and growth factors include, but arepreferably not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF,EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic,FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C),GDNF, G-CSF, GM-CSF, GF-β1, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.),IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF),Inhibin α, Inhibin (3, IP-10, keratinocyte growth factor-2 (KGF-2), KGF,Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocytecolony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.),MDC (69 a.a.), MIG, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloidprogenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growthfactor, β-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB,PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stem cell factor (SCF), TARC,TGF-α, TGF-β2, TGF-β3, tumour necrosis factor (TNF), TNF-α, TNF receptorI, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2,VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER 1, HER2, HER 3 and HER 4, CD4, human chemokine receptors CXCR4 or CCR5,non-structural protein type 3 (NS3) from the hepatitis C virus,TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB, influenza,Hepatitis E, MMP-12, internalizing receptors that are over-expressed oncertain cells, such as the epidermal growth factor receptor (EGFR),ErBb2 receptor on tumor cells, an internalising cellular receptor, LDLreceptor, FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGFreceptor, VEGF receptor, PsmAr, an extracellular matrix protein,elastin, fibronectin, laminin, α1-antitrypsin, tissue factor proteaseinhibitor, PDK1, GSK1, Bad, caspase-9, Forkhead, an antigen ofHelicobacter pylori, an antigen of Mycobacterium tuberculosis, and anantigen of influenza virus. It will be appreciated that this list is byno means exhaustive.

In one embodiment of the invention, the variable domains are derivedfrom a respective antibody directed against the antigen or epitope. In apreferred embodiment the variable domains are derived from a repertoireof single variable antibody domains.

In a further aspect, the present invention provides one or more nucleicacid molecules encoding at least a dual-specific ligand as hereindefined. The dual specific ligand may be encoded on a single nucleicacid molecule; alternatively, each domain may be encoded by a separatenucleic acid molecule. Where the ligand is encoded by a single nucleicacid molecule, the domains may be expressed as a fusion polypeptide, inthe manner of a scFv molecule, or may be separately expressed andsubsequently linked together, for example using chemical linking agents.Ligands expressed from separate nucleic acids will be linked together byappropriate means.

The nucleic acid may further encode a signal sequence for export of thepolypeptides from a host cell upon expression and may be fused with asurface component of a filamentous bacteriophage particle (or othercomponent of a selection display system) upon expression.

In a further aspect the present invention provides a vector comprisingnucleic acid encoding a dual specific ligand according to the presentinvention.

In a yet further aspect, the present invention provides a host celltransfected with a vector encoding a dual specific ligand according tothe present invention.

Expression from such a vector may be configured to produce, for exampleon the surface of a bacteriophage particle, variable domains forselection. This allows selection of displayed variable domains and thusselection of ‘dual-specific ligands’ using the method of the presentinvention.

The present invention further provides a kit comprising at least adual-specific ligand according to the present invention.

Dual-Specific ligands according to the present invention preferablycomprise combinations of heavy and light chain domains. For example, thedual specific ligand may comprise a V_(H) domain and a V_(L) domain,which may be linked together in the form of an scFv. In addition, theligands may comprise one or more C_(H) or C_(L) domains. For example,the ligands may comprise a C_(H)1 domain, C_(H)2 or C_(H)3 domain,and/or a CL domain, Cμ1, Cμ2, Cμ3 or Cμ4 domains, or any combinationthereof. A hinge region domain may also be included. Such combinationsof domains may, for example, mimic natural antibodies, such as IgG orIgM, or fragments thereof, such as Fv, scFv, Fab or F(ab′)₂ molecules.Other structures, such as a single arm of an IgG molecule comprisingV_(H), V_(L), C_(H)1 and C_(L) domains, are envisaged.

In a preferred embodiment of the invention, the variable regions areselected from single domain V gene repertoires. Generally the repertoireof single antibody domains is displayed on the surface of filamentousbacteriophage. In a preferred embodiment each single antibody domain isselected by binding of a phage repertoire to antigen.

In a preferred embodiment of the invention each single variable domainmay be selected for binding to its target antigen or epitope in theabsence of a complementary variable region. In an alternativeembodiment, the single variable domains may be selected for binding toits target antigen or epitope in the presence of a complementaryvariable region. Thus, the first single variable domain may be selectedin the presence of a third complementary variable domain, and the secondvariable domain may be selected in the presence of a fourthcomplementary variable domain. The complementary third or fourthvariable domain may be the natural cognate variable domain having thesame specificity as the single domain being tested, or a non-cognatecomplementary domain—such as a “dummy” variable domain.

Preferably, the dual specific ligand of the invention comprises only twovariable domains although several such ligands may be incorporatedtogether into the same protein, for example two such ligands can beincorporated into an IgG or a multimeric immunoglobulin, such as IgM.Alternatively, in another embodiment a plurality of dual specificligands are combined to form a multimer. For example, two different dualspecific ligands are combined to create a tetra-specific molecule.

It will be appreciated by one skilled in the art that the light andheavy variable domains of a dual-specific ligand produced according tothe method of the present invention may be on the same polypeptidechain, or alternatively, on different polypeptide chains. In the casethat the variable domains are on different polypeptide chains, then theymay be linked via a linker, generally a flexible linker (such as apolypeptide chain), a chemical linking group, or any other method knownin the art.

In a further aspect, the present invention provides a compositioncomprising a dual-specific ligand, obtainable by a method of the presentinvention, and a pharmaceutically acceptable carrier, diluent orexcipient.

Moreover, the present invention provides a method for the treatmentand/or prevention of disease using a ‘dual-specific ligand’ or acomposition according to the present invention.

In a second configuration, the present invention provides multispecificligands which comprise at least two non-complementary variable domains.For example, the ligands may comprise a pair of V_(H) domains or a pairof V_(L) domains. Advantageously, the domains are of non-camelid origin;preferably they are human domains or comprise human framework regions(FWs) and one or more heterologous CDRs. CDRs and framework regions arethose regions of an immunoglobulin variable domain as defined in theKabat database of Sequences of Proteins of Immunological Interest.

Preferred human framework regions are those encoded by germ line genesegments DP47 and DPK9. Advantageously, FW1, FW2 and FW3 of a V_(H) orV_(L) domain have the sequence of FW1, FW2 or FW3 from DP47 or DPK9. Thehuman frameworks may optionally contain mutations, for example up toabout 5 amino acid changes or up to about 10 amino acid changescollectively in the human frameworks used in the ligands of theinvention.

The variable domains in the multispecific ligands according to thesecond configuration of the invention may be arranged in an open or aclosed conformation; that is, they may be arranged such that thevariable domains can bind their cognate ligands independently andsimultaneously, or such that only one of the variable domains may bindits cognate ligand at any one time.

The inventors have realised that under certain structural conditions,non-complementary variable domains (for example two light chain variabledomains or two heavy chain variable domains) may be present in a ligandsuch that binding of a first epitope to a first variable domain inhibitsthe binding of a second epitope to a second variable domain, even thoughsuch non-complementary domains do not operate together as a cognatepair.

Advantageously, the ligand comprises two or more pairs of variabledomains; that is, it comprises at least four variable domains.Advantageously, the four variable domains comprise frameworks of humanorigin.

In a preferred embodiment, the human frameworks are identical to thoseof human germ line sequences.

The present inventors consider that such antibodies will be ofparticular use in ligand binding assays for therapeutic and other uses.

Thus, in a first aspect of the second configuration, the presentinvention provides a method for producing a multispecific ligandcomprising the steps of:

a) selecting a first epitope binding domain by its ability to bind to afirst epitope,b) selecting a second epitope binding domain by its ability to bind to asecond epitope,c) combining the epitope binding domains; andd) selecting the closed conformation multispecific ligand by its abilityto bind to said first second epitope and said second epitope.

In a further aspect of the second configuration, the invention providesmethod for preparing a closed conformation multi-specific ligandcomprising a first epitope binding domain having a first epitope bindingspecificity and a non-complementary second epitope binding domain havinga second epitope binding specificity, wherein the first and secondbinding specificities compete for epitope binding such that the closedconformation multi-specific ligand may not bind both epitopessimultaneously, said method comprising the steps of:

a) selecting a first epitope binding domain by its ability to bind to afirst epitope,b) selecting a second epitope binding domain by its ability to bind to asecond epitope,c) combining the epitope binding domains such that the domains are in aclosed conformation; andd) selecting the closed conformation multispecific ligand by its abilityto bind to said first second epitope and said second epitope, but not toboth said first and second epitopes simultaneously.

Moreover, the invention provides a closed conformation multi-specificligand comprising a first epitope binding domain having a first epitopebinding specificity and a non-complementary second epitope bindingdomain having a second epitope binding specificity, wherein the firstand second binding specificities compete for epitope binding such thatthe closed conformation multi-specific ligand may not bind both epitopessimultaneously.

An alternative embodiment of the above aspect of the of the secondconfiguration of the invention optionally comprises a further step (b1)comprising selecting a third or further epitope binding domain. In thisway the multi-specific ligand produced, whether of open or closedconformation, comprises more than two epitope binding specificities. Ina preferred aspect of the second configuration of the invention, wherethe multi-specific ligand comprises more than two epitope bindingdomains, at least two of said domains are in a closed conformation andcompete for binding; other domains may compete for binding or may befree to associate independently with their cognate epitope(s).

According to the present invention the term ‘multi-specific ligand’refers to a ligand which possesses more than one epitope bindingspecificity as herein defined.

As herein defined the term ‘closed conformation’ (multi-specific ligand)means that the epitope binding domains of the ligand are attached to orassociated with each other, optionally by means of a protein skeleton,such that epitope binding by one epitope binding domain competes withepitope binding by another epitope binding domain. That is, cognateepitopes may be bound by each epitope binding domain individually, butnot simultaneously. The closed conformation of the ligand can beachieved using methods herein described.

“Open conformation” means that the epitope binding domains of the ligandare attached to or associated with each other, optionally by means of aprotein skeleton, such that epitope binding by one epitope bindingdomain does not compete with epitope binding by another epitope bindingdomain.

As referred to herein, the term ‘competes’ means that the binding of afirst epitope to its cognate epitope binding domain is inhibited when asecond epitope is bound to its cognate epitope binding domain. Forexample, binding may be inhibited sterically, for example by physicalblocking of a binding domain or by alteration of the structure orenvironment of a binding domain such that its affinity or avidity for anepitope is reduced.

In a further embodiment of the second configuration of the invention,the epitopes may displace each other on binding. For example, a firstepitope may be present on an antigen which, on binding to its cognatefirst binding domain, causes steric hindrance of a second bindingdomain, or a conformational change therein, which displaces the epitopebound to the second binding domain.

Advantageously, binding is reduced by 25% or more, advantageously 40%,50%, 60%, 70%, 80%, 90% or more, and preferably up to 100% or nearly so,such that binding is completely inhibited. Binding of epitopes can bemeasured by conventional antigen binding assays, such as ELISA, byfluorescence based techniques, including FRET, or by techniques such assurface plasmon resonance which measure the mass of molecules. Specificbinding of an antigen-binding protein to an antigen or epitope can bedetermined by a suitable assay, including, for example, Scatchardanalysis and/or competitive binding assays, such as radioimmunoassays(RIA), enzyme immunoassays such as ELISA and sandwich competitionassays, and the different variants thereof.

Binding affinity is preferably determined using surface plasmonresonance (SPR) and the Biacore (Karlsson et al., 1991), using a Biacoresystem (Uppsala, Sweden). The Biacore system uses surface plasmonresonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton andMyszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecularinteractions in real time, and uses surface plasmon resonance which candetect changes in the resonance angle of light at the surface of a thingold film on a glass support as a result of changes in the refrativeindex of the surface up to 300 nm away. Biacore analysis convenientlygenerates association rate constants, dissociation rate constants,equilibrium dissociation constants, and affinity constants. Bindingaffinity is obtained by assessing the association and dissociation rateconstants using a Biacore surface plasmon resonance system (Biacore,Inc.). A biosensor chip is activated for covalent coupling of the targetaccording to the manufacturer's (Biacore) instructions. The target isthen diluted and injected over the chip to obtain a signal in responseunits of immobilized material. Since the signal in resonance units (RU)is proportional to the mass of immobilized material, this represents arange of immobilized target densities on the matrix. Dissociation dataare fit to a one-site model to obtain k_(off)+/−s.d. (standard deviationof measurements). Pseudo-first order rate constant (Kd's) are calculatedfor each association curve, and plotted as a function of proteinconcentration to obtain k_(on)+/−s.e. (standard error of fit).Equilibrium dissociation constants for binding, Kd's, are calculatedfrom SPR measurements as k_(off)/k_(on).

As described by Phizicky and Field in Microb. Rev. (1995) 59:114-115, asuitable antigen, such as HSA, is immobilized on a dextran polymer, anda solution containing a ligand for HSA, such as a single variabledomain, flows through a cell, contacting the immobilized HSA. The singlevariable domain retained by immobilized HSA alters the resonance angleof impinging light, resulting in a change in refractive index broughtabout by increased amounts of protein, i.e. the single variable domain,near the dextran polymer. Since all proteins have the same refractiveindex and since there is a linear correlation between resonance angleshift and protein concentration near the surface, changes in the proteinconcentration at the surface due to protein/protein binding can bemeasured, see Phizicky and Field, supra. To determine a bindingconstant, the increase in resonance units (RU) is measured as a functionof time by passing a solution of single variable domain protein past theimmobilized ligand (HSA) until the RU values stabilize, then thedecrease in RU is measured as a function of time with buffer lacking thesingle variable domain. This procedure is repeated at several differentconcentrations of single variable domain protein. Detailed theoreticalbackground and procedures are described by R. Karlsson, et. al. (991) J.Immunol Methods, 145, 229.

The instrument software produces an equilibrium disociation constant(Kd) as described above. An equilibrium disociation constant determinedthrough the use of Surface plasmon resonance is described in U.S. Pat.No. 5,573,957, as being based on a table of dR_(A)/dt and R_(A) values,where R in this example is the HSA/single variable domain complex asmeasured by the Biacore in resonance units and where dR/dt is the rateof formation of HSA/single variable domain complexes, i.e. thederivative of the binding curve; plotting the graph dR_(A)/dt vs R_(A)for several different concentrations of single variable domain, andsubsequently plotting the slopes of these lines vs. the concentration ofsingle variable domain, the slope of this second graph being theassociation rate constant (M⁻¹, s⁻¹). The Dissociation Rate Constant orthe rate at which the HSA and the single variable domain release fromeach other, can be determined utilizing the dissociation curve generatedon the Biacore. By plotting and determining the slope of the log of thedrop in the response vs time curve, the dissociation rate constant canbe measured. The Equilibrium disociation constant Kd=Dissociation RateConstant/Association Rate Constant.

According to the method of the present invention, advantageously, eachepitope binding single variable domain is of a different epitope bindingspecificity.

In the context of the present invention, first and second “epitopes” areunderstood to be epitopes which are not the same and are not bound by asingle monospecific ligand. They may be on different antigens or on thesame antigen, but separated by a sufficient distance that they do notform a single entity that could be bound by a single mono-specificV_(H)/V_(L) binding pair of a conventional antibody. Experimentally, ifboth of the individual variable domains in single chain antibody form(domain antibodies or dAbs) are separately competed by a monospecificV_(H)/V_(L) ligand against two epitopes then those two epitopes are notsufficiently far apart to be considered separate epitopes according tothe present invention.

The closed conformation multispecific ligands of the invention do notinclude ligands as described in WO 02/02773. Thus, the ligands of thepresent invention do not comprise complementary V_(H)/V_(L) Pairs whichbind any one or more antigens or epitopes co-operatively. Instead, theligands according to the invention preferably comprise non-complementaryV_(H)-V_(H) or V_(L)-V_(L) pairs. Advantageously, each V_(H) or V_(L)domain in each V_(H)-V_(H) or V_(L)-V_(L) pair has a different epitopebinding specificity, and the epitope binding sites are so arranged thatthe binding of an epitope at one site competes with the binding of anepitope at another site.

According to the present invention, advantageously, each epitope bindingdomain comprises an immunoglobulin variable domain. More advantageously,each epitope binding domain will be either a variable light chain domain(V_(L)) or a variable heavy chain domain (V_(H)) of an antibody. In thesecond configuration of the present invention, the immunoglobulindomains when present on a ligand according to the present invention arenon-complementary, that is they do not associate to form a V_(H)/V_(L)antigen binding site. Thus, multi-specific ligands as defined in thesecond configuration of the invention comprise immunoglobulin domains ofthe same sub-type, that is either variable light chain domains (V_(L))or variable heavy chain domains (V_(H)). Moreover, where the ligandaccording to the invention is in the closed conformation, theimmunoglobulin domains may be of the camelid V_(HH) type.

In an alternative embodiment, the ligand(s) according to the inventiondo not comprise a camelid V_(HH) domain. More particularly, theligand(s) of the invention do not comprise one or more amino acidresidues that are specific to camelid V_(HH) domains as compared tohuman V_(H) domains.

Advantageously, the single variable domains are derived from antibodiesselected for binding activity against different antigens or epitopes.For example, the variable domains may be isolated at least in part byhuman immunisation. Alternative methods are known in the art, includingisolation from human antibody libraries and synthesis of artificialantibody genes.

In selected embodiments a single variable domain is a naturallyoccurring single variable domain. In other selected embodiments thesingle variable domain is non-naturally occurring. The term “naturallyoccurring” is used herein to indicate that an object, e.g., a proteindomain, e.g., a single variable domain, or antibody single variabledomain, can be found in nature. Thus, a naturally occurring proteindomain, such as a V region of an antibody, exists in a protein, e.g. inan antibody chain protein, expressed in nature, for example, in anon-recombinant species, e.g., mammals, primates, rodents, fish, birds,reptiles, etc. For the avoidance of doubt, a single variable domainisolated from a repertoire of polypeptides expressed from nucleic acidsto which diversity was introduced in vitro is a non-naturally occurringsingle variable domain. For the further avoidance of doubt, an antibodysingle variable domain originating from an antibody resulting fromimmunization of an animal is a naturally-occurring single variabledomain.

The variable domains advantageously bind superantigens, such as proteinA or protein L. Binding to superantigens is a property of correctlyfolded antibody variable domains, and allows such domains to be isolatedfrom, for example, libraries of recombinant or mutant domains.

Epitope binding domains according to the present invention comprise aprotein scaffold and epitope interaction sites (which are advantageouslyon the surface of the protein scaffold).

Epitope binding domains may also be based on protein scaffolds orskeletons other than immunoglobulin domains. For example, naturalbacterial receptors such as SpA have been used as scaffolds for thegrafting of CDRs to generate ligands which bind specifically to one ormore epitopes. Details of this procedure are described in U.S. Pat. No.5,831,012. Other suitable scaffolds include those based on fibronectinand affibodies. Details of suitable procedures are described in WO98/58965. Other suitable scaffolds include lipocallin and CTLA4, asdescribed in van den Beuken et al., J. Mol. Biol. (2001) 310, 591-601,and scaffolds such as those described in WO0069907 (Medical ResearchCouncil), which are based for example on the ring structure of bacterialGroEL or other chaperone polypeptides.

Protein scaffolds may be combined; for example, CDRs may be grafted onto a CTLA4 scaffold and used together with immunoglobulin V_(H) or V_(L)domains to form a multivalent ligand. Likewise, fibronectin, lipocallinand other scaffolds may be combined.

It will be appreciated by one skilled in the art that the epitopebinding domains of a closed conformation multispecific ligand producedaccording to the method of the present invention may be on the samepolypeptide chain, or alternatively, on different polypeptide chains. Inthe case that the variable domains are on different polypeptide chains,then they may be linked via a linker, advantageously a flexible linker(such as a polypeptide chain), a chemical linking group, or any othermethod known in the art.

The first and the second epitope binding domains may be associatedeither covalently or non-covalently. In the case that the domains arecovalently associated, then the association may be mediated for exampleby disulphide bonds.

In the second configuration of the invention, the first and the secondepitopes are preferably different. They may be, or be part of,polypeptides, proteins or nucleic acids, which may be naturallyoccurring or synthetic. In this respect, the ligand of the invention maybind an epitope or antigen and act as an antagonist or agonist (e.g.,EPO receptor agonist). The epitope binding domains of the ligand in oneembodiment have the same epitope specificity, and may for examplesimultaneously bind their epitope when multiple copies of the epitopeare present on the same antigen. In another embodiment, these epitopesare provided on different antigens such that the ligand can bind theepitopes and bridge the antigens. One skilled in the art will appreciatethat the choice of epitopes and antigens is large and varied. They maybe for instance human or animal proteins, cytokines, cytokine receptors,enzymes co-factors for enzymes or DNA binding proteins. Suitablecytokines and growth factors include but are preferably not limited to:ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor, ENA-78,Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic, fibroblastgrowth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF,GF-β1, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11,IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β,IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF,Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitoryfactor, monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.),MIG, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitorfactor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3,NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α,SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β2, TGF-β3,tumour necrosis factor (TNF), TNF-α, TNF receptor 1, TNF receptor II,TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER 1, HER 2, HER 3, HER 4,TACE recognition site, TNF BP-I and TNF BP-II, CD4, human chemokinereceptors CXCR4 or CCR5, non-structural protein type 3 (NS3) from thehepatitis C virus, TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori,TB, influenza, Hepatitis E, MMP-12, internalising receptors areover-expressed on certain cells, such as the epidermal growth factorreceptor (EGFR), ErBb2 receptor on tumor cells, an internalisingcellular receptor, LDL receptor, FGF2 receptor, ErbB2 receptor,transferrin receptor, PDGF receptor, VEGF receptor, PsmAr, anextracellular matrix protein, elastin, fibronectin, laminin,al-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1, Bad,caspase-9, Forkhead, an of an antigen of Helicobacter pylori, an antigenof Mycobacterium tuberculosis, and an antigen of influenza virus, aswell as any target disclosed in Annex 2 or Annex 3 hereto, whether incombination as set forth in the Annexes, in a different combination orindividually. Cytokine receptors include receptors for the abovecytokines, e.g. IL-1 R1; IL-6R; IL-10R; IL-18R, as well as receptors forcytokines set forth in Annex 2 or Annex 3 and also receptors disclosedin Annex 2 and 3. It will be appreciated that this list is by no meansexhaustive. Where the multispecific ligand binds to two epitopes (on thesame or different antigens), the antigen(s) may be selected from thislist.

Advantageously, dual specific ligands may be used to target cytokinesand other molecules which cooperate synergistically in therapeuticsituations in the body of an organism. The invention therefore providesa method for synergising the activity of two or more cytokines,comprising administering a dual specific ligand capable of binding tosaid two or more cytokines. In this aspect of the invention, the dualspecific ligand may be any dual specific ligand, including a ligandcomposed of complementary and/or non-complementary domains, a ligand inan open conformation, and a ligand in a closed conformation. Forexample, this aspect of the invention relates to combinations of V_(H)domains and V_(L) domains, V_(H) domains only and V_(L) domains only.

Synergy in a therapeutic context may be achieved in a number of ways.For example, target combinations may be therapeutically active only ifboth targets are targeted by the ligand, whereas targeting one targetalone is not therapeutically effective. In another embodiment, onetarget alone may provide some low or minimal therapeutic effect, buttogether with a second target the combination provides a synergisticincrease in therapeutic effect.

Preferably, the cytokines bound by the dual specific ligands of thisaspect of the invention are selected from the list shown in Annex 2.

Moreover, dual specific ligands may be used in oncology applications,where one specificity targets CD89, which is expressed by cytotoxiccells, and the other is tumour specific. Examples of tumour antigenswhich may be targeted are given in Annex 3.

In one embodiment of the second configuration of the invention, thevariable domains are derived from an antibody directed against the firstand/or second antigen or epitope. In a preferred embodiment the variabledomains are derived from a repertoire of single variable antibodydomains. In one example, the repertoire is a repertoire that is notcreated in an animal or a synthetic repertoire. In another example, thesingle variable domains are not isolated (at least in part) by animalimmunisation. Thus, the single domains can be isolated from a naïvelibrary.

The second configuration of the invention, in another aspect, provides amulti-specific ligand comprising a first epitope binding domain having afirst epitope binding specificity and a non-complementary second epitopebinding domain having a second epitope binding specificity. The firstand second binding specificities may be the same or different.

In a further aspect, the present invention provides a closedconformation multi-specific ligand comprising a first epitope bindingdomain having a first epitope binding specificity and anon-complementary second epitope binding domain having a second epitopebinding specificity wherein the first and second binding specificitiesare capable of competing for epitope binding such that the closedconformation multi-specific ligand cannot bind both epitopessimultaneously.

In a still further aspect, the invention provides open conformationligands comprising non-complementary binding domains, wherein thedomains are specific for a different epitope on the same target. Suchligands bind to targets with increased avidity. Similarly, the inventionprovides multivalent ligands comprising non-complementary bindingdomains specific for the same epitope and directed to targets whichcomprise multiple copies of said epitope, such as IL-5, PDGF-AA,PDGF-BB, TGF-β2, TGF-β3 and TNFα, for example, as well as human TNFReceptor 1 and human TNFα.

In a similar aspect, ligands according to the invention can beconfigured to bind individual epitopes with low affinity, such thatbinding to individual epitopes is not therapeutically significant; butthe increased avidity resulting from binding to two epitopes provides atherapeutic benefit. In a particular example, epitopes may be targetedwhich are present individually on normal cell types, but presenttogether only on abnormal or diseased cells, such as tumour cells. Insuch a situation, only the abnormal or diseased cells are effectivelytargeted by the bispecific ligands according to the invention.

Ligand specific for multiple copies of the same epitope, or adjacentepitopes, on the same target (known as chelating dAbs) may also betrimeric or polymeric (tertrameric or more) ligands comprising three,four or more non-complementary binding domains. For example, ligands maybe constructed comprising three or four V_(H) domains or V_(L) domains.

Moreover, ligands are provided which bind to multisubunit targets,wherein each binding domain is specific for a subunit of said target.The ligand may be dimeric, trimeric or polymeric.

Preferably, the multi-specific ligands according to the above aspects ofthe invention are obtainable by the method of the first aspect of theinvention.

According to the above aspect of the second configuration of theinvention, advantageously the first epitope binding domain and thesecond epitope binding domains are non-complementary immunoglobulinvariable domains, as herein defined. That is either V_(H)-V_(H) orV_(L)-V_(L) variable domains.

Chelating dAbs in particular may be prepared according to a preferredaspect of the invention, namely the use of anchor dAbs, in which alibrary of dimeric, trimeric or multimeric dAbs is constructed using avector which comprises a constant dAb upstream or downstream of a linkersequence, with a repertoire of second, third and further dAbs beinginserted on the other side of the linker. For example, the anchor orguiding dAb may be TAR1-5 (V_(κ)), TAR1-27(V_(κ)), TAR2h-5(VH) orTAR2h-6(V_(κ)).

In alternative methodologies, the use of linkers may be avoided, forexample by the use of non-covalent bonding or natural affinity betweenbinding domains such as V_(H) and V_(κ). The invention accordinglyprovides a method for preparing a chelating multimeric ligand comprisingthe steps of:

(a) providing a vector comprising a nucleic acid sequence encoding asingle binding domain specific for a first epitope on a target;(b) providing a vector encoding a repertoire comprising second bindingdomains specific for a second epitope on said target, which epitope canbe the same or different to the first epitope, said second epitope beingadjacent to said first epitope; and(c) expressing said first and second binding domains; and(d) isolating those combinations of first and second binding domainswhich combine together to produce a target-binding dimer.

The first and second epitopes are adjacent such that a multimeric ligandis capable of binding to both epitopes simultaneously. This provides theligand with the advantages of increased avidity if binding. Where theepitopes are the same, the increased avidity is obtained by the presenceof multiple copies of the epitope on the target, allowing at least twocopies to be simultaneously bound in order to obtain the increasedavidity effect.

The binding domains may be associated by several methods, as well as theuse of linkers. For example, the binding domains may comprise cysresidues, avidin and streptavidin groups or other means for non-covalentattachment post-synthesis; those combinations which bind to the targetefficiently will be isolated. Alternatively, a linker may be presentbetween the first and second binding domains, which are expressed as asingle polypeptide from a single vector, which comprises the firstbinding domain, the linker and a repertoire of second binding domains,for instance as described above.

In a preferred aspect, the first and second binding domains associatenaturally when bound to antigen; for example, V_(H) and V_(L) (e.g.V_(κ)) domains, when bound to adjacent epitopes, will naturallyassociate in a three-way interaction to form a stable dimer. Suchassociated proteins can be isolated in a target binding assay. Anadvantage of this procedure is that only binding domains which bind toclosely adjacent epitopes, in the correct conformation, will associateand thus be isolated as a result of their increased avidity for thetarget.

In an alternative embodiment of the above aspect of the secondconfiguration of the invention, at least one epitope binding domaincomprises a non-immunoglobulin ‘protein scaffold’ or ‘protein skeleton’as herein defined. Suitable non-immunoglobulin protein scaffolds includebut are preferably not limited to any of those selected from the groupconsisting of: SpA, fibronectin, GroEL and other chaperones, lipocallin,CCTLA4 and affibodies, as set forth above.

According to the above aspect of the second configuration of theinvention, advantageously, the epitope binding domains are attached to a‘protein skeleton’. Advantageously, a protein skeleton according to theinvention is an immunoglobulin skeleton.

According to the present invention, the term ‘immunoglobulin skeleton’refers to a protein which comprises at least one immunoglobulin fold andwhich acts as a nucleus for one or more epitope binding domains, asdefined herein.

Preferred immunoglobulin skeletons as herein defined includes any one ormore of those selected from the following: an immunoglobulin moleculecomprising at least (i) the CL (kappa or lambda subclass) domain of anantibody; or (ii) the CH1 domain of an antibody heavy chain; animmunoglobulin molecule comprising the CH1 and CH2 domains of anantibody heavy chain; an immunoglobulin molecule comprising the CH1, CH2and CH3 domains of an antibody heavy chain; or any of the subset (ii) inconjunction with the CL (kappa or lambda subclass) domain of anantibody. A hinge region domain may also be included. Such combinationsof domains may, for example, mimic natural antibodies, such as IgG orIgM, or fragments thereof, such as Fv, scFv, Fab or F(ab′)₂ molecules.Those skilled in the art will be aware that this list is not intended tobe exhaustive.

Linking of the skeleton to the epitope binding domains, as hereindefined may be achieved at the polypeptide level, that is afterexpression of the nucleic acid encoding the skeleton and/or the epitopebinding domains. Alternatively, the linking step may be performed at thenucleic acid level. Methods of linking a protein skeleton according tothe present invention, to the one or more epitope binding domainsinclude the use of protein chemistry and/or molecular biology techniqueswhich will be familiar to those skilled in the art and are describedherein.

Advantageously, the closed conformation multispecific ligand maycomprise a first domain capable of binding a target molecule, and asecond domain capable of binding a molecule or group which extends thehalf-life of the ligand. For example, the molecule or group may be abulky agent, such as HSA or a cell matrix protein. As used herein, thephrase “molecule or group which extends the half-life of a ligand”refers to a molecule or chemical group which, when bound by adual-specific ligand as described herein increases the in vivo half-lifeof such dual specific ligand when administered to an animal, relative toa ligand that does not bind that molecule or group. Examples ofmolecules or groups that extend the half-life of a ligand are describedherein below. In a preferred embodiment, the closed conformationmultispecific ligand may be capable of binding the target molecule onlyon displacement of the half-life enhancing molecule or group. Thus, forexample, a closed conformation multispecific ligand is maintained incirculation in the bloodstream of a subject by a bulky molecule such asHSA. When a target molecule is encountered, competition between thebinding domains of the closed conformation multispecific ligand resultsin displacement of the HSA and binding of the target.

A ligand according to any aspect of the present invention, incudes aligand having or consisting of at least one single variable domain, inthe form of a monomer single variable domain or in the form of multiplesingle variable domains, i.e. a multimer. The ligand can be modified tocontain additional moities, such as a fusion protein, or a conjugate.Such a multimeric ligand, e.g., in the form of a dual specific ligand,and/or such a ligand comprising or consisting of a single variabledomain, i.e. a dAb monomer useful in constructing such a multimericligand, may advantageously dissociate from their cognate target(s) witha Kd of 300 nM or less, 300 nM to 5 pM (i.e., 3×10⁻⁷ to 5×10⁻¹²M),preferably 50 nM to 20 pM, or 5 nM to 200 pM or 1 nM to 100 pM, 1×10⁻⁷ Mor less, 1×10⁻⁸ M or less, 1×10⁻⁹ M or less, 1×10⁻¹⁰ M or less, 1×10⁻¹¹M or less; and/or a K_(off) rate constant of 5×10⁻¹ to 1×10⁻⁷ S⁻¹,preferably 1×10⁻² to 1×10⁻⁶ S⁻¹, or 5×10⁻³ to 1×10⁻⁵ S⁻¹, or 5×10⁻¹ S⁻¹or less, or 1×10⁻² S⁻¹ or less, or 1×10⁻³ S⁻¹ or less, or 1×10⁻⁴ S⁻¹ orless, or 1×10⁻⁵ S⁻¹ or less, or 1×10⁻⁶ S⁻¹ or less as determined, forexample, by surface plasmon resonance. The Kd rate constant is definedas K_(off)/K_(on). A Kd value greater than Molar is generally consideredto indicate non-specific binding. Preferably, a single variable domainwill specifically bind a target antigen or epitope with an affinity ofless than 500 nM, preferably less than 200 nM, and more preferably lessthan 10 nM, such as less than 500 pM

In particular the invention provides an anti-TNFα dAb monomer (or dualspecific ligand comprising such a dAb), homodimer, heterodimer orhomotrimer ligand, wherein each dAb binds TNFα. The ligand binds to TNFαwith a Kd of 300 nM to 5 pM (i.e., 3×10⁻⁷ to 5×10⁻¹²M), preferably 50 nMto 20 pM, more preferably 5 nM to 200 pM and most preferably 1 nM to 100pM; expressed in an alternative manner, the Kd is 1×10⁻⁷ M or less,preferably 1×10⁻⁸ M or less, more preferably 1×10⁻⁹ M or less,advantageously 1×10⁻¹⁰ M or less and most preferably 1×10⁻¹¹ M or less;and/or a K_(off) rate constant of 5×10⁻¹ to 1×10⁻⁷ S⁻¹, preferably1×10⁻² to 1×10⁻⁶ S⁻¹ more preferably 5×10⁻³ to 1×10⁻⁵ S⁻¹, for example5×10⁻¹ S⁻¹ or less, preferably 1×10⁻² S⁻¹ or less, more preferably1×10⁻³ S⁻¹ or less, advantageously 1×10⁻⁴ S⁻¹ or less, furtheradvantageously 1×10⁻⁵ S⁻¹ or less, and most preferably 1×10⁻⁶ S⁻¹ orless, as determined by surface plasmon resonance.

Preferably, the ligand neutralises TNFα in a standard L929 assay with anND50 of 500 nM to 50 pM, preferably or 100 nM to 50 pM, advantageously10 nM to 100 pM, more preferably 1 nM to 100 pM; for example 50 nM orless, preferably 5 nM or less, advantageously 500 pM or less, morepreferably 200 pM or less and most preferably 100 pM or less.

Preferably, the ligand inhibits binding of TNF alpha to TNF alphaReceptor I (p55 receptor) with an IC50 of 500 nM to 50 pM, preferably100 nM to 50 pM, more preferably 10 nM to 100 pM, advantageously 1 nM to100 pM; for example 50 nM or less, preferably 5 nM or less, morepreferably 500 pM or less, advantageously 200 pM or less, and mostpreferably 100 pM or less. Preferably, the TNFα is Human TNFα.

Furthermore, the invention provides an anti-TNF Receptor I dAb monomer,or dual specific ligand comprising such a dAb, that binds to TNFReceptor I with a Kd of 300 nM to 5 pM (i.e., 3×10⁻⁷ to 5×10⁻¹²M),preferably 50 nM to 20 pM, more preferably 5 nM to 200 pM and mostpreferably 1 nM to 100 pM, for example 1×10⁻⁷ M or less, preferably1×10⁻⁸ M or less, more preferably 1×10⁻⁹ M or less, advantageously1×10⁻¹⁰ M or less and most preferably 1×10⁻¹¹ M or less; and/or aK_(off) rate constant of 5×10⁻¹ to 1×10⁻⁷ S⁻¹, preferably 1×10⁻² to1×10⁻⁶ S⁻¹, more preferably 5×10⁻³ to 1×10⁻⁵ S⁻¹, for example 5×10⁻¹ S⁻¹or less, preferably 1×10⁻² S⁻¹ or less, advantageously 1×10⁻³ S⁻¹ orless, more preferably 1×10⁻⁴ S⁻¹ or less, still more preferably 1×10⁻⁵S⁻¹ or less, and most preferably 1×10⁻⁶ S⁻¹ or less, preferably asdetermined by surface plasmon resonance.

Preferably, the dAb monomer ligand neutralises TNFα in a standard assay(e.g., the L929 or HeLa assays described herein) with an ND50 of 500 nMto 50 pM, preferably 100 nM to 50 pM, more preferably 10 nM to 100 pM,advantageously 1 nM to 100 pM; for example 50 nM or less, preferably 5nM or less, more preferably 500 pM or less, advantageously 200 pM orless, and most preferably 100 pM or less.

Preferably, the dAb monomer or ligand inhibits binding of TNF alpha toTNF alpha Receptor I (p55 receptor) with an IC50 of 500 nM to 50 pM,preferably 100 nM to 50 pM, more preferably 10 nM to 100 pM,advantageously 1 nM to 100 pM; for example 50 nM or less, preferably 5nM or less, more preferably 500 pM or less, advantageously 200 pM orless, and most preferably 100 pM or less. Preferably, the TNF Receptor Itarget is Human TNFα.

Furthermore, the invention provides a dAb monomer (or dual specificligand comprising such a dAb) that binds to serum albumin (SA) with a Kdof 1 nM to 50004 (i.e., 1×10⁻⁹ to 5×10⁻⁴), preferably 100 nM to 1004.Preferably, for a dual specific ligand comprising a first anti-SA dAband a second dAb to another target, the affinity (e.g. Kd and/or K_(off)as measured by surface plasmon resonance, e.g. using Biacore) of thesecond dAb for its target is from 1 to 100000 times (preferably 100 to100000, more preferably 1000 to 100000, or 10000 to 100000 times) theaffinity of the first dAb for SA. For example, the first dAb binds SAwith an affinity of approximately 10 μM, while the second dAb binds itstarget with an affinity of 100 pM. Preferably, the serum albumin ishuman serum albumin (HSA).

In one embodiment, the first dAb (or a dAb monomer) binds SA (e.g., HSA)with a Kd of approximately 50, preferably 70, and more preferably 100,150 or 200 nM.

The invention moreover provides dimers, trimers and polymers of theaforementioned dAb monomers, in accordance with the above aspect of thepresent invention.

Ligands according to the invention, including dAb monomers, dimers andtrimers, can be linked to an antibody Fc region, comprising one or bothof C_(H)2 and C_(H)3 domains, and optionally a hinge region. Forexample, vectors encoding ligands linked as a single nucleotide sequenceto an Fc region may be used to prepare such polypeptides.

In a further aspect of the second configuration of the invention, thepresent invention provides one or more nucleic acid molecules encodingat least a multispecific ligand as herein defined. In one embodiment,the multispecific ligand is a closed conformation ligand. In anotherembodiment, it is an open conformation ligand. The multispecific ligandmay be encoded on a single nucleic acid molecule; alternatively, eachepitope binding domain may be encoded by a separate nucleic acidmolecule. Where the multispecific ligand is encoded by a single nucleicacid molecule, the domains may be expressed as a fusion polypeptide, ormay be separately expressed and subsequently linked together, forexample using chemical linking agents. Ligands expressed from separatenucleic acids will be linked together by appropriate means.

The nucleic acid may further encode a signal sequence for export of thepolypeptides from a host cell upon expression and may be fused with asurface component of a filamentous bacteriophage particle (or othercomponent of a selection display system) upon expression. Leadersequences, which may be used in bacterial expression and/or phage orphagemid display, include pelB, stII, ompA, phoA, bla and pelA.

In a further aspect of the second configuration of the invention thepresent invention provides a vector comprising nucleic acid according tothe present invention.

In a yet further aspect, the present invention provides a host celltransfected with a vector according to the present invention.

Expression from such a vector may be configured to produce, for exampleon the surface of a bacteriophage particle, epitope binding domains forselection. This allows selection of displayed domains and thus selectionof ‘multispecific ligands’ using the method of the present invention.

In a preferred embodiment of the second configuration of the invention,the epitope binding domains are immunoglobulin variable domains and areselected from single domain V gene repertoires. Generally the repertoireof single antibody domains is displayed on the surface of filamentousbacteriophage. In a preferred embodiment each single antibody domain isselected by binding of a phage repertoire to antigen.

The present invention further provides a kit comprising at least amultispecific ligand according to the present invention, which may be anopen conformation or closed conformation ligand. Kits according to theinvention may be, for example, diagnostic kits, therapeutic kits, kitsfor the detection of chemical or biological species, and the like.

In a further aspect still of the second configuration of the invention,the present invention provides a homogenous immunoassay using a ligandaccording to the present invention.

In a further aspect still of the second configuration of the invention,the present invention provides a composition comprising a closedconformation multispecific ligand, obtainable by a method of the presentinvention, and a pharmaceutically acceptable carrier, diluent orexcipient.

Moreover, the present invention provides a method for the treatment ofdisease using a ‘closed conformation multispecific ligand’ or acomposition according to the present invention.

In a preferred embodiment of the invention the disease is cancer or aninflammatory disease, e.g. rheumatoid arthritis, asthma or Crohn'sdisease.

In a further aspect of the second configuration of the invention, thepresent invention provides a method for the diagnosis, includingdiagnosis of disease using a closed conformation multispecific ligand,or a composition according to the present invention. Thus in general thebinding of an analyte to a closed conformation multispecific ligand maybe exploited to displace an agent, which leads to the generation of asignal on displacement. For example, binding of analyte (second antigen)could displace an enzyme (first antigen) bound to the antibody providingthe basis for an immunoassay, especially if the enzyme were held to theantibody through its active site.

Thus in a final aspect of the second configuration, the presentinvention provides a method for detecting the presence of a targetmolecule, comprising:

(a) providing a closed conformation multispecific ligand bound to anagent, said ligand being specific for the target molecule and the agent,wherein the agent which is bound by the ligand leads to the generationof a detectable signal on displacement from the ligand;(b) exposing the closed conformation multispecific ligand to the targetmolecule; and(c) detecting the signal generated as a result of the displacement ofthe agent.

According to the above aspect of the second configuration of theinvention, advantageously, the agent is an enzyme, which is inactivewhen bound by the closed conformation multi-specific ligand.Alternatively, the agent may be any one or more selected from the groupconsisting of the following: the substrate for an enzyme, and afluorescent, luminescent or chromogenic molecule which is inactive orquenched when bound by the ligand.

Sequences similar or homologous (e.g., at least about 70% sequenceidentity) to the sequences disclosed herein are also part of theinvention. In some embodiments, the sequence identity at the amino acidlevel can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or higher. At the nucleic acid level, the sequence identity canbe about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or higher. Alternatively, substantial identity exists when thenucleic acid segments will hybridize under selective hybridizationconditions (e.g., very high stringency hybridization conditions), to thecomplement of the strand. The nucleic acids may be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form.

The percent identity can refer to the percent identity along the entirestretch of the length of the amino acid or nucleotide sequence. Whenspecified, the percent identity of the amino acid or nucleic acidsequence refers to the percent identity to sequence(s) from one or morediscrete regions of the referenced amino acid or nucleic acid sequence,for instance, along one or more antibody CDR regions, and/or along oneor more antibody variable framework regions. For example, the sequenceidentity at the amino acid level across one or more CDRs of an antibodyheavy or light chain single variable domain can have about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity tothe amino acid sequence of corresponding CDRs of an antibody heavy orlight chain single variable domain, respectively. At the nucleic acidlevel, the nucleic acid sequence encoding one or more CDRs of anantibody heavy or light chain single variable domain can have at least70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orhigher, identity to the nucleic acid sequence encoding the correspondingCDRs of an antibody heavy or light chain single variable domain. At thenucleic acid level, the nucleic acid sequence encoding one CDR of anantibody heavy or light chain single variable domain can have a percentidentity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or higher, than the nucleic acid sequence encoding thecorresponding CDR of an antibody heavy or light chain single variabledomain, respectively. In some embodiments, the structural characteristicof percent identity is coupled to a functional aspect. For instance, insome embodiments, a nucleic acid sequence or amino acid sequence withless than 100% identity to a referenced nucleic acid or amino acidsequence is also required to display at least one functional aspect ofthe reference amino acid sequence or of the amino acid sequence encodedby the referenced nucleic acid. In other embodiments, a nucleic acidsequence or amino acid sequence with less than 100% identity to areferenced nucleic acid or amino acid sequence, respectively, is alsorequired to display at least one functional aspect of the referenceamino acid sequence or of the amino acid sequence encoded by thereferenced nucleic acid, but that functional characteristic can beslightly altered, e.g., confer an increased affinity to a specifiedantigen relative to that of the reference.

Calculations of “homology” or “sequence identity” or “similarity”between two sequences (the terms are used interchangeably herein) areperformed as follows. The sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “homology” is equivalent to aminoacid or nucleic acid “identity”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

Advantageously, the BLAST algorithm (version 2.0) is employed forsequence alignment, with parameters set to default values. The BLASTalgorithm is described in detail at the world wide web site (“www”) ofthe National Center for Biotechnology Information (“NCBI”) of theNational Institutes of Health (“NIH”) of the U.S. government (“gov”), inthe “/Blast/” directory, in the “blast_help.html” file. The searchparameters are defined as follows, and are advantageously set to thedefined default parameters.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci.USA 87(6):2264-8 (see the “blast_help.html” file, as described above)with a few enhancements. The BLAST programs were tailored for sequencesimilarity searching, for example to identify homologues to a querysequence. The programs are not generally useful for motif-stylesearching. For a discussion of basic issues in similarity searching ofsequence databases, see Altschul et al. (1994).

The five BLAST programs available at the National Center forBiotechnology Information web site perform the following tasks:

“blastp” compares an amino acid query sequence against a proteinsequence database;“blastn” compares a nucleotide query sequence against a nucleotidesequence database;“blastx” compares the six-frame conceptual translation products of anucleotide query sequence (both strands) against a protein sequencedatabase;“tblastn” compares a protein query sequence against a nucleotidesequence database dynamically translated in all six reading frames (bothstrands).“tblastx” compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

BLAST uses the following search parameters:

HISTOGRAM Display a histogram of scores for each search; default is yes.(See parameter H in the BLAST Manual).

DESCRIPTIONS Restricts the number of short descriptions of matchingsequences reported to the number specified; default limit is 100descriptions. (See parameter V in the manual page). See also EXPECT andCUTOFF.

ALIGNMENTS Restricts database sequences to the number specified forwhich high-scoring segment pairs (HSPs) are reported; the default limitis 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

EXPECT The statistical significance threshold for reporting matchesagainst database sequences; the default value is 10, such that 10matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

CUTOFF Cutoff score for reporting high-scoring segment pairs. Thedefault value is calculated from the EXPECT value (see above). HSPs arereported for a database sequence only if the statistical significanceascribed to them is at least as high as would be ascribed to a lone HSPhaving a score equal to the CUTOFF value. Higher CUTOFF values are morestringent, leading to fewer chance matches being reported. (Seeparameter S in the BLAST Manual). Typically, significance thresholds canbe more intuitively managed using EXPECT.

MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTNand TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992,Proc. Natl. Aacad. Sci. USA 89(22):10915-9). The valid alternativechoices include: PAM40, PAM120, PAM250 and IDENTITY. No alternatescoring matrices are available for BLASTN; specifying the MATRIXdirective in BLASTN requests returns an error response.

STRAND Restrict a TBLASTN search to just the top or bottom strand of thedatabase sequences; or restrict a BLASTN, BLASTX or TBLASTX search tojust reading frames on the top or bottom strand of the query sequence.

FILTER Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993) Computers and Chemistry 17:149-163, or segmentsconsisting of short-periodicity internal repeats, as determined by theXNU program of Claverie & States, 1993, Computers and Chemistry17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman(see the world wide web site of the NCBI). Filtering can eliminatestatistically significant but biologically uninteresting reports fromthe blast output (e.g., hits against common acidic-, basic- orproline-rich regions), leaving the more biologically interesting regionsof the query sequence available for specific matching against databasesequences.

Low complexity sequence found by a filter program is substituted usingthe letter “N” in nucleotide sequence (e.g., “N” repeated 13 times) andthe letter “X” in protein sequences (e.g., “X” repeated 9 times).

Filtering is only applied to the query sequence (or its translationproducts), not to database sequences. Default filtering is DUST forBLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both,when applied to sequences in SWISS-PROT, so filtering should not beexpected to always yield an effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect.

NCBI-gi Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

Most preferably, sequence comparisons are conducted using the simpleBLAST search algorithm provided at the NCBI world wide web sitedescribed above, in the “/BLAST” directory.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the diversification of V_(H)/HSA at positions H50, H52,H52a, H53, H55, H56, H58, H95, H96, H97, H98 (DVT or NNK encodedrespectively) which are in the antigen binding site of V_(H) HSA. Thesequence of V_(κ) is diversified at positions L50, L53.

FIG. 2 shows Library 1: Germ line V_(κ)/DVT V_(H),

-   -   Library 2: Germ line V_(κ)/NNK V_(H),    -   Library 3: Germ line V_(H)/DVT V_(κ)    -   Library 4: Germ line V_(H)/NNK V_(κ)

In phage display/ScFv format. These libraries were pre-selected forbinding to generic ligands protein A and protein L so that the majorityof the clones and selected libraries are functional. Libraries wereselected on HSA (first round) and β-gal (second round) or HSA β-galselection or on β-gal (first round) and HSA (second round) β-gal HSAselection. Soluble scFv from these clones of PCR are amplified in thesequence. One clone encoding a dual specific antibody K8 was chosen forfurther work.

FIG. 3 shows an alignment of V_(H) chains and V_(κ) chains.

FIG. 4 shows the characterisation of the binding properties of the K8antibody, the binding properties of the K8 antibody characterised bymonoclonal phage ELISA, the dual specific K8 antibody was found to bindHSA and β-gal and displayed on the surface of the phage with absorbentsignals greater than 1.0. No cross reactivity with other proteins wasdetected.

FIG. 5 shows soluble scFv ELISA performed using known concentrations ofthe K8 antibody fragment. A 96-well plate was coated with 100 μg of HSA,BSA and β-gal at 10 μg/ml and 100 μg/ml of Protein A at 1 μg/mlconcentration. 50 μg of the serial dilutions of the K8 scFv was appliedand the bound antibody fragments were detected with Protein L-HRP. ELISAresults confirm the dual specific nature of the K8 antibody.

FIG. 6 shows the binding characteristics of the clone K8V_(κ)/dummyV_(H) analysed using soluble scFv ELISA. Production of the soluble scFvfragments was induced by IPTG as described by Harrison et al, MethodsEnzymol. 1996; 267:83-109 and the supernatant containing scFv assayeddirectly. Soluble scFv ELISA is performed as described in example 1 andthe bound scFvs were detected with Protein L-HRP. The ELISA resultsrevealed that this clone was still able to bind β-gal, whereas bindingBSA was abolished.

FIG. 7 shows the sequence of variable domain vectors 1 and 2.

FIG. 8 is a map of the CH vector used to construct a V_(H)1/V_(H)2multipsecific ligand.

FIG. 9 is a map of the V_(κ) vector used to construct a V_(κ)1/V_(κ)2multispecific ligand.

FIG. 10 TNF receptor assay comparing TAR1-5 dimer 4, TAR1-5-19 dimer 4and TAR1-5-19 monomer.

FIG. 11 TNF receptor assay comparing TAR1-5 dimers 1-6. All dimers havebeen FPLC purified and the results for the optimal dimeric species areshown.

FIG. 12 TNF receptor assay of TAR1-5 19 homodimers in different formats:dAb-linker-dAb format with 3U, 5U or 7U linker, Fab format and cysteinehinge linker format.

FIG. 13 Dummy VH sequence for library 1. The sequence of the V_(H)framework based on germ line sequence DP47-JH4b. Positions where NNKrandomisation (N=A or T or C or G nucleotides; K=G or T nucleotides) hasbeen incorporated into library 1 are indicated in bold underlined text.

FIG. 14 Dummy VH sequence for library 2. The sequence of the V_(H)framework based on germ line sequence DP47-JH4b. Positions where NNKrandomisation (N=A or T or C or G nucleotides; K=G or T nucleotides) hasbeen incorporated into library 2 are indicated in bold underlined text.

FIG. 15 Dummy V_(κ) sequence for library 3. The sequence of the V_(κ)framework based on germ line sequence DP_(κ)9-J_(κ)1. Positions whereNNK randomisation (N=A or T or C or G nucleotides; K=G or T nucleotides)has been incorporated into library 3 are indicated in bold underlinedtext.

FIG. 16 Nucleotide and amino acid sequence of anti MSA dAbs MSA 16 andMSA 26.

FIG. 17 Inhibition Biacore of MSA 16 and 26. Purified dAbs MSA16 andMSA26 were analysed by inhibition Biacore to determine Kd. Briefly, thedAbs were tested to determine the concentration of dAb required toachieve 200 RUs of response on a Biacore CM5 chip coated with a highdensity of MSA. Once the required concentrations of dAb had beendetermined, MSA antigen at a range of concentrations around the expectedKd was premixed with the dAb and incubated overnight. Binding to the MSAcoated Biacore chip of dAb in each of the premixes was then measured ata high flow-rate of 30 μl/minute.

FIG. 18 Serum levels of MSA16 following injection. Serum half life ofthe dAb MSA16 was determined in mouse. MSA16 was dosed as single i.v.injections at approx 1.5 mg/kg into CD1 mice. Modelling with a 2compartment model showed MSA16 had a t1/2α of 0.98 hr, a t1/2β of 36.5hr and an AUC of 913 hr·mg/ml. MSA16 had a considerably lengthened halflife compared with HEL4 (an anti-hen egg white lysozyme dAb) which had at1/2α of 0.06 hr and a t1/2β of 0.34 hr.

FIG. 19 ELISA (a) and TNF receptor assay (c) showing inhibition of TNFbinding 30 with a Fab-like fragment comprising MSA26Ck and TARI-5-19CH.The TNF receptor assay (b) was conducted in the presence of a constantconcentration of heterodimer (18 nM) and a dilution series of MSA andHSA. Addition of MSA with the Fab-like fragment reduces the level ofinhibition. An ELISA plate coated with 1 p,g/ml TNFα was probed withdual specific VK CH and VK CK Fab like fragment and also with a controlTNFα binding dAb at a concentration calculated to give a similar signalon the ELISA. Both the dual specific and control dAb were used to probethe ELISA plate in the presence and in the absence of 2 mg/ml MSA. Thesignal in the dual specific well 5 was reduced by more than 50% but thesignal in the dAb well was not reduced at all (see FIG. 19a ). The samedual specific protein was also put into the receptor assay with andwithout MSA and competition by MSA was also shown (see FIG. 19c ). Thisdemonstrates that binding of MSA to the dual specific is competitivewith binding to TNFα.

FIG. 20 TNF receptor assay showing inhibition of TNF binding with adisulphide bonded heterodimer of TAR1-5-19 dAb and MSA16 dAb. Additionof MSA with the dimer reduces the level of inhibition in a dosedependant manner. The TNF receptor assay (FIG. 19 (b)) was conducted inthe presence of a constant concentration of heterodimer (18 nM) and adilution series of MSA and HSA. The presence of HSA at a range ofconcentrations (up to 2 mg/ml) did not cause a reduction in the abilityof the dimer to inhibit TNFα. However, the addition of MSA caused a dosedependant reduction in the ability of the dimer to inhibit TNFα (FIG.19a ). This demonstrates that MSA and TNFα compete for binding to thecys bonded TAR1-5-19, MSA16 dimer. MSA and HSA alone did not have aneffect on the TNF binding level in the assay.

FIG. 21 Purified recombinant domains of human serum albumin (HSA), lanes1-3 contain HSA domains I, II and III, respectively.

FIG. 22 Example of an immunoprecipitation showing that an HSA-bindingdAb binds full length HSA (lane 8) and HSA domain II (lane 6), but doesnot bind HSA domains I and III (lanes 5 and 7, respectively). Anon-HSA-binding dAb does not pull down either full length HSA or HSAdomains I, II, or III (lanes 1-4).

FIG. 23. Example of identification of HSA domain binding by a dAb asidentified by surface plasmon resonance. The dAb under study wasinjected as described onto a low density coated human serum albumin CM5sensor chip (Biacore). At point 1, the dAb under study was injectedalone at 1 μM. At point 2, using the co-inject command, sample injectionwas switched to a mixture of 1 μM dAb plus 7 μM HSA domain 1, 2 or 3,produced in Pichia. At point 3, sample injection was stopped, and bufferflow continued. Results for two different dAbs are shown in 23 A and 23B. When the dAb is injected with the HSA domain that it binds, it formsa complex that can no longer bind the HSA on the chip, hence the Biacoresignal drops at point 2, with an off-rate that reflects the 3-wayequilibrium between dAb, soluble HSA domain, and chip bound HSA. Whenthe domain does not bind the dAb, the signal remains unchanged at point2, and starts to drop only at point 3, where flow is switched to buffer.In both these cases, the dAb binds HSA domain 2.

FIG. 24 Antibody sequences of AlbudAb™ (a dAb which specifically bindsserum albumin) clones identified by phage selection. All clones havebeen aligned to the human germ line genes. Residues that are identicalto germ line have been represented by ‘.’. In the VH CDR3, the symbol‘-’ has been used to facilitate alignment but does not represent aresidue. All clones were selected from libraries based on a single humanframework comprising the heavy-chain germ line genes V3-23/DP47 and JH4bfor the VH libraries and the κ light chain genes O12/O2/DPK9 and J_(κ)1for the V_(κ) libraries with side chain diversity incorporated atpositions in the antigen binding site.

FIG. 25 Alignments of the three domains of human serum albumin. Theconservation of the cysteine residues can clearly be seen.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Complementary” Two immunoglobulin domains are “complementary” wherethey belong to families of structures which form cognate pairs or groupsor are derived from such families and retain this feature. For example,a V_(H) domain and a V_(L) domain of an antibody are complementary; twoV_(H) domains are not complementary, and two V_(L) domains are notcomplementary. Complementary domains may be found in other members ofthe immunoglobulin superfamily, such as the V_(α) and V_(β) (or γ and β)domains of the T-cell receptor. In the context of the secondconfiguration of the present invention, non-complementary domains do notbind a target molecule cooperatively, but act independently on differenttarget epitopes which may be on the same or different molecules. Domainswhich are artificial, such as domains based on protein scaffolds whichdo not bind epitopes unless engineered to do so, are non-complementary.Likewise, two domains based on (for example) an immunoglobulin domainand a fibronectin domain are not complementary.

“Immunoglobulin” This refers to a family of polypeptides which retainthe immunoglobulin fold characteristic of antibody molecules, whichcontains two β sheets and, usually, a conserved disulphide bond. Membersof the immunoglobulin superfamily are involved in many aspects ofcellular and non-cellular interactions in vivo, including widespreadroles in the immune system (for example, antibodies, T-cell receptormolecules and the like), involvement in cell adhesion (for example theICAM molecules) and intracellular signalling (for example, receptormolecules, such as the PDGF receptor). The present invention isapplicable to all immunoglobulin superfamily molecules which possessbinding domains. Preferably, the present invention relates toantibodies.

“Combining” Variable domains according to the invention are combined toform a group of domains; for example, complementary domains may becombined, such as V_(L) domains being combined with V_(H) domains.Non-complementary domains may also be combined. Domains may be combinedin a number of ways, involving linkage of the domains by covalent ornon-covalent means.

“Domain” A domain is a folded protein structure which retains itstertiary structure independently of the rest of the protein. Generally,domains are responsible for discrete functional properties of proteins,and in many cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain.

As used herein, a “single variable domain” is a domain which canspecifically bind an epitope, an antigen or a ligand independently, thatis, without the requirement for another binding domain to co-operativelybind the epitope, antigen or ligand. Such an epitope, antigen or ligandcan be naturally occurring, or can be a modification of a naturaloccurring epitope, antigen or ligand, or can be synthetic. The“variable” portion of the single variable domain essentially determinesthe binding specificity of each particular single variable domain. Thus,the term “variable” in the context of single variable domains, refers tothe fact that the sequence variability is not evenly distributed througha single variable domain, but is essentially distributed between theframework or skeleton portions of the single variable domain. Forexample, in an antibody single variable domain, the variability isconcentrated in one to three segments commonly known as complementaritydetermining regions (CDRs). The one or more CDRs can be distributedbetween antibody framework regions (FR) of a light chain or of a heavychain to form either an antibody light chain single variable domain oran antibody heavy chain single variable domain, respectively, each ofwhich specifically binds an epitope independently of another bindingdomain. Similarly structured is a T-cell receptor single variabledomain, with its one to three CDRs distributed between the TCR frameworkdomains.

Thus, the variable portions conferring the binding specificity of singlevariable domains may differ extensively in sequence from other singlevariable domains having substantially the same remaining scaffoldportion, and accordingly, may have a diverse range of bindingspecificities. Scaffolds of single variable domains include antibodyframework scaffolds, consensus antibody frameworks, and scaffoldsoriginating and/or derived from bacterial proteins, e.g. GroEL, GroEs,SpA, SpG, and from eukaryotic proteins, e.g., CTLA-4, lipocallins,fibronectin, etc. One source of the variable portions of single variabledomains include one or more CDRs, which can be grafted ontonon-immunoglobulin scaffolds as well as antibody framework scaffolds togenerate antibody single variable domains. Another source of variationin a single variable domain can be the diversification of chosenpositions in a non-immunoglobulin framework scaffold such asfibronectin, to generate single variable domains, using molecularbiology techniques, such as NNK codon diversity. Similarly, this sourceof variation is also applicable to an antibody single variable domain.

An antibody single variable domain can be derived from antibodysequences encoded and/or generated by an antibody producing species, andincludes fragment(s) and/or derivatives of the antibody variable region,including one or more framework regions, or framework consensussequences, and/or one or more CDRs. Accordingly, an antibody singlevariable domain includes fragment(s) and/or derivative(s) of an antibodylight chain variable region, or of an antibody heavy chain variableregion, or of an antibody VHH region. For example, antibody VHH regionsinclude those that are endogenous to camelids: e.g., camels and llamas,and the new antigen receptor (NAR) from nurse and wobbegong sharks (Rouxet al., 1998 PNAS 95(20):11804-9) and the VH region from spotted raffish(Rast et al., 1998 Immunogenetics 47:234-245). Antibody light chainvariable domains and antibody heavy chain variable domains include thoseendogenous to an animal species including, but preferably not limitedto, human, mouse, rat, porcine, cynomolgus, hamster, horse, cow, goat,dog, cat, and avian species, e.g. human VKappa and human VH3,respectively. Antibody light chain variable regions and antibody heavychain variable regions, also includes consensus antibody frameworks, asdescribed infra, including those of V region families, such as the VH3family. A T-cell receptor single variable domain is a single variabledomain which is derived from a T-cell receptor chain(s), e.g., α, β, γand β□ chains, and which binds an epitope or an antigen or a ligandindependently of another binding domain for that epitope, antigen orligand, analogously to antibody single variable domains.

An antibody single variable domain also encompasses a protein domainwhich comprises a scaffold which is not derived from an antibody or aT-cell receptor, and which has been genetically engineered to displaydiversity in binding specificity relative to its pre-engineered state,by incorporating into the scaffold, one or more of a CDR1, a CDR2 and/ora CDR3, derivative or fragment thereof, or an entire antibody V domain.An antibody single variable domain can also include bothnon-immunoglobulin scaffold and immunoglobulin scaffolds as illustratedby the GroEL single variable domain multimers described infra.Preferably the CDR(s) is from an antibody V domain of an antibody chain,e.g., VH, VL, and VHH. The antibody chain can be one which specificallybinds an antigen or epitope in concert with a second antibody chain, orthe antibody chain can be one which specifically binds an antigen orepitope independently of a second antibody chain, such as VHH chain. Theintegration of one or more CDRs into an antibody single variable domainwhich comprises a non-immunoglobulin scaffold must result in the nonimmunoglobulin scaffold's single variable domain's ability tospecifically bind an epitope or an antigen or a ligand independently ofanother binding domain for that epitope, antigen or ligand.

A single domain is transformed into a single variable domain byintroducing diversity at the site(s) designed to become the bindingsite, followed by selection for desired binding characteristics using,for example, display technologies. Diversity can be introduced inspecific sites of a non-immunoglobulin scaffold of interest byrandomizing the amino acid sequence of specific loops of the scaffold,e.g. by introducing NNK codons. This mechanism of generating diversityfollowed by selection of desired binding characteristics is similar tothe natural selection of high affinity, antigen-specific antibodiesresulting from diversity generated in the loops which make up theantibody binding site in nature. Ideally, a single domain which is smalland contains a fold similar to that of an antibody loop, is transformedinto a single variable domain, variants of the single variable domainare expressed, from which single variable domains containing desiredbinding specificities and characteristics can be selected from librariescontaining a large number of variants of the single variable domain.

Nomenclature of single variable domains: sometimes the nomenclature ofan antibody single variable domain is abbreviated by leaving off thefirst “d” or the letters “Dom”, for example, Ab7h24 is identical todAb7h24 which is identical to DOM7h24.

By antibody single variable domain is meant a folded polypeptide domaincomprising sequences characteristic of antibody variable domains. Ittherefore includes complete antibody variable domains and modifiedvariable domains, for example, in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least in part the binding activity andspecificity of the full-length domain.

“Repertoire” A collection of diverse variants, for example polypeptidevariants which differ in their primary sequence. A library used in thepresent invention will encompass a repertoire of polypeptides comprisingat least 1000 members.

“Library” The term library refers to a mixture of heterogeneouspolypeptides or nucleic acids. The library is composed of members, eachof which have a single polypeptide or nucleic acid sequence. To thisextent, library is synonymous with repertoire. Sequence differencesbetween library members are responsible for the diversity present in thelibrary. The library may take the form of a simple mixture ofpolypeptides or nucleic acids, or may be in the form of organisms orcells, for example bacteria, viruses, animal or plant cells and thelike, transformed with a library of nucleic acids. Preferably, eachindividual organism or cell contains only one or a limited number oflibrary members. Advantageously, the nucleic acids are incorporated intoexpression vectors, in order to allow expression of the polypeptidesencoded by the nucleic acids. In a preferred aspect, therefore, alibrary may take the form of a population of host organisms, eachorganism containing one or more copies of an expression vectorcontaining a single member of the library in nucleic acid form which canbe expressed to produce its corresponding polypeptide member. Thus, thepopulation of host organisms has the potential to encode a largerepertoire of genetically diverse polypeptide variants.

A “closed conformation multi-specific ligand” describes a multi-specificligand as herein defined comprising at least two epitope binding domainsas herein defined. The term ‘closed conformation’ (multi-specificligand) means that the epitope binding domains of the ligand arearranged such that epitope binding by one epitope binding domaincompetes with epitope binding by another epitope binding domain. Thatis, cognate epitopes may be bound by each epitope binding domainindividually but not simultaneously. The closed conformation of theligand can be achieved using methods herein described.

“Antibody” An antibody (for example IgG, IgM, IgA, IgD or IgE) orfragment (such as a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, closedconformation multispecific antibody, disulphide-linked scFv, diabody)whether derived from any species naturally producing an antibody, orcreated by recombinant DNA technology; whether isolated from serum,B-cells, hybridomas, transfectomas, yeast or bacteria).

“Dual-specific ligand” A ligand comprising a first immunoglobulin singlevariable domain and a second immunoglobulin single variable domain asherein defined, wherein the variable domains are capable of binding totwo different antigens or two epitopes on the same antigen which are notnormally bound by a monospecific immunoglobulin. For example, the twoepitopes may be on the same hapten, but are not the same epitope orsufficiently adjacent to be bound by a monospecific ligand. The dualspecific ligands according to the invention are composed of variabledomains which have different specificities, and do not contain mutuallycomplementary variable domain pairs which have the same specificity.Thus, dual specific ligands, which as defined herein contain two singlevariable domains, are a subset of multimeric ligands, which as definedherein contain two or more single variable domains, wherein at least twoof the single variable domains are capable of binding to two differentantigens or to two different epitopes on the same antigen. Further, adual specific ligand as defined herein is also distinct from a ligandcomprising an antibody single variable domain, and a second antigenand/or epitope binding domain which is not a single variable domain.Further still, a dual specific ligand as defined herein is also distinctform a ligand containing a first and a second antigen/epitope bindingdomain, where neither antigen/epitope binding domain is a singlevariable domain as defined herein.

“Antigen” A molecule that is bound by a ligand according to the presentinvention. Typically, antigens are bound by antibody ligands and arecapable of raising an antibody response in vivo. It may be apolypeptide, protein, nucleic acid or other molecule. Generally, thedual specific ligands according to the invention are selected for targetspecificity against a particular antigen. In the case of conventionalantibodies and fragments thereof, the antibody binding site defined bythe variable loops (L1, L2, L3 and H1, H2, H3) is capable of binding tothe antigen.

“Epitope” A unit of structure conventionally bound by an immunoglobulinpair. Epitopes define the minimum binding site for an antibody, and thusrepresent the target of specificity of an antibody. In the case of asingle domain antibody, an epitope represents the unit of structurebound by a variable domain in isolation. An epitope binding domaincomprises a protein scaffold and epitope interaction sites (which areadvantageously on the surface of the protein scaffold). An epitopebinding domain can comprise epitope interaction sites that arenonlinear, e.g. where the epitope binding domain comprises multipleepitope interaction sites that have intervening regions between them,e.g., CDRs separated by FRs, or are present on separate polypeptidechains. Alternatively, an epitope binding domain can comprise a linearepitope interaction site composed of contiguously encoded amino acids onone polypeptide chain.

“Generic ligand” A ligand that binds to all members of a repertoire.Generally, not bound through the antigen binding site as defined above.Non-limiting examples include protein A, protein L and protein G.

“Selecting” Derived by screening, or derived by a Darwinian selectionprocess, in which binding interactions are made between a domain and theantigen or epitope or between an antibody and an antigen or epitope.Thus a first variable domain may be selected for binding to an antigenor epitope in the presence or in the absence of a complementary variabledomain.

“Universal framework” A single antibody framework sequence correspondingto the regions of an antibody conserved in sequence as defined by Kabat(“Sequences of Proteins of Immunological Interest”, US Department ofHealth and Human Services) or corresponding to the human germ lineimmunoglobulin repertoire or structure as defined by Chothia and Lesk,(1987) J. Mol. Biol. 196:910-917. The invention provides for the use ofa single framework, or a set of such frameworks, which has been found topermit the derivation of virtually any binding specificity thoughvariation in the hypervariable regions alone.

As used herein “conjugate” refers to a composition comprising an antigenbinding fragment of an antibody that binds serum albumin that is bondedto a drug.

As used herein, the term “small molecule” means a compound having amolecular weight of less than 1,500 daltons, preferably less than 1000daltons.

Such conjugates include “drug conjugates,” which comprise anantigen-binding fragment of an antibody that binds serum albumin towhich a drug is covalently bonded, and “noncovlaent drug conjugates,”which comprise an antigen-binding fragment of an antibody that bindsserum albumin to which a drug is noncovalently bonded.

As used herein, “drug conjugate” refers to a composition comprising anantigen-binding fragment of an antibody that binds serum albumin towhich a drug is covalently bonded. The drug can be covalently bonded tothe antigen-binding fragment directly or indirectly through a suitablelinker moiety. The drug can be bonded to the antigen-binding fragment atany suitable position, such as the amino-terminus, the carboxyl-terminusor through suitable amino acid side chains (e.g., the amino group oflysine).

“Half-life” The time taken for the serum concentration of the ligand toreduce by 50%, in vivo, for example due to degradation of the ligandand/or clearance or sequestration of the ligand by natural mechanisms.The ligands of the invention are stabilised in vivo and their half-lifeincreased by binding to molecules which resist degradation and/orclearance or sequestration. Typically, such molecules are naturallyoccurring proteins which themselves have a long half-life in vivo. Thehalf-life of a ligand is increased if its functional activity persists,in vivo, for a longer period than a similar ligand which is not specificfor the half-life increasing molecule. Thus, a ligand specific for HSAand a target molecule is compared with the same ligand wherein thespecificity for HSA is not present, that it does not bind HSA but bindsanother molecule. For example, it may bind a second epitope on thetarget molecule. Typically, the half life is increased by 10%, 20%, 30%,40%, 50% or more. Increases in the range of 2×, 3×, 4×, 5×, 10×, 20×,30×, 40×, 50× or more of the half life are possible. Alternatively, orin addition, increases in the range of up to 30×, 40×, 50×, 60×, 70×,80×, 90×, 100×, 150× of the half life are possible.

The phrase “substantially the same” when used to compare the T beta halflife of a ligand with the T beta half life of serum albumin in a hostmeans that the T beta half life of the ligand in a host varies no morethan 50% from the T beta half life of serum albumin itself in the samehost, preferably a human host, e.g., the T beta half life of such aligand is no more than 50% less or no more than 50% greater than the Tbeta half life of serum albumin in a specified host. Preferably, whenreferring to the phrase “substantially the same”, the T beta half lifeof the ligand in a host varies no more than 20% to 10% from the halflife of serum albumin itself, and more preferably, varies no more than9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or less from the half life ofserum albumin itself, or does not vary at all from the half life ofserum albumin itself.

Alternatively, the phrase “not substantially the same” when used tocompare the T beta half life of a ligand with the T beta half life ofserum albumin in a host means that the T beta half life of the ligand ina host varies at least 50% from the T beta half life of serum albuminitself in the same host, preferably a human host, e.g., the T beta halflife of the ligand is more than 50% greater than the T beta half life ofserum albumin in a specified host.

“Homogeneous immunoassay” An immunoassay in which analyte is detectedwithout need for a step of separating bound and un-bound reagents.

“Substantially identical” or “substantially homologous” A first aminoacid or nucleotide sequence that contains a sufficient number ofidentical or equivalent (e.g., with a similar side chain, e.g.,conserved amino acid substitutions) amino acid residues or nucleotidesto a second amino acid or nucleotide sequence such that the first andsecond amino acid or nucleotide sequences have similar activities. Inthe case of first and second antibodies and/or single variable domainsdescribed herein, the second antibody or single variable domain has thesame binding specificity as the first and has at least 50%, or at leastup to 55%, 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% ofthe affinity of the first antibody or single variable domain.

As used herein, the terms “low stringency,” “medium stringency,” “highstringency,” or “very high stringency conditions” describe conditionsfor nucleic acid hybridization and washing. Guidance for performinghybridization reactions can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated herein by reference in its entirety. Aqueous and nonaqueousmethods are described in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and preferably (4) very high stringencyhybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C.,followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very highstringency conditions (4) are the preferred conditions and the ones thatshould be used unless otherwise specified.

“Surface Plasmon Resonance” Competition assays can be used to determineif a specific antigen or epitope, such as human serum albumin, competeswith another antigen or epitope, such as cynomolgus serum albumin, forbinding to a serum albumin binding ligand described herein, such as aspecific dAb. Similarly competition assays can be used to determine if afirst ligand such as dAb, competes with a second ligand such as a dAbfor binding to a target antigen or epitope. The term “competes” as usedherein refers to substance, such as a molecule, compound, preferably aprotein, which is able to interfere to any extent with the specificbinding interaction between two or more molecules. The phrase “does notcompetitively inhibit” means that substance, such as a molecule,compound, preferably a protein, does not interfere to any measurable orsignificant extent with the specific binding interaction between two ormore molecules. The specific binding interaction between two or moremolecules preferably includes the specific binding interaction between asingle variable domain and its cognate partner or target. Theinterfering or competing molecule can be another single variable domainor it can be a molecule that that is structurally and/or functionallysimilar to a cognate partner or target.

A single variable domain includes an immunoglobulin single variabledomain and a non-immunoglobulin single variable domain which containsone, two, three or more CDR regions from an immunoglobulin variabledomain, such as an antibody variable domain, including an antibody heavyor antibody light chain single variable domain. The single variabledomain can be derived from an animal, including a human, rat, mouse,pig, monkey, camelidae, such as an antibody variable (V) region, or itcan be derived from a microorganism such as E. coli in the case of thenon-immunglobulin scaffold of GroEL and GroEs. A single variable domaincan be partially or totally artificial, or can be generated usingrecombinant molecular biology technology.

In vitro competition assays for determining the ability of a singlevariable domain to compete for binding to a target to another targetbinding domain, such as another single variable domain, as well as fordeterming the Kd, are well known in the art. One preferred competitionassay is a surface plasmon resonance assay, which has the advantages ofbeing fast, sensitive and useful over a wide range of proteinconcentrations, and requiring small amounts of sample material. Apreferred surface plasmon resonance assay competition is a competitionBiacore experiment. A competition Biacore experiment can be used todetermine whether, for example, cynomolgus serum albumin and human serumalbumin compete for binding to a ligand such as dAb DOM7h-x. Oneexperimental protocol for such an example is as follows.

For example, after coating a CM5 sensor chip (Biacore AB) at 25° C. withapproximately 1000 resonance units (RUs) of human serum albumin (HSA), apurified dAb is injected over the antigen surface at a singleconcentration (e.g., 1 um) alone, and in combination with a dilutionseries of the cynomolgus serum albumin (CSA). The serial dilutions ofHSA were mixed with a constant concentration (40 nM) of the purifieddAb. A suitable dilution series of CSA would be starting at 5 uM CSA,with six two-fold dilutions down to 78 nM CSA. These solutions must beallowed to reach equilibrium before injection. Following the injection,a response reading was taken to measure the resulting binding RUs forthe dAb alone and each of the several dAb/CSA mixtures, the data beingused in accordance with BIA evaluation software, generate adose-response curve for each CSA's inhibition of the AlbudAb™'s (a dAbwhich specifically binds serum albumin) binding to the chip on which HSAis immobilized. By comparing the bound RUs of dAb alone with the boundRUs of dAb+CSA, one will be able to see whether the CSA competes withthe HSA to bind the dAb. If it does compete, then as the CSAconcentration in solution is increased, the RUs of dAb bound to HSA willdecrease. If there is no competition, then adding CSA will have noimpact on how much dAb binds to HSA.

One of skill would know how to adapt this or other protocols in order toperform this competition assay on a variety of different ligands,including the several ligands described herein that bind serum albumin.The variety of ligands includes, but is not limited to, monomer singlevariable domains, including single variable domains comprising animmunoglobulin and/or a non-immunoglobulin scaffold, dAbs, dual specificligands, and multimers of these ligands. One of skill would also knowhow to adapt this protocol in order to compare the binding of severaldifferent pairs of antigens and/or epitopes to a ligand using thiscompetition assay.

These competition experiments can provide a numeric cut-off by which onecan determine if an antigen or epitope competes with another antigen orepitope for binding to a specific ligand, preferably a dAb. For example,in the experiment outlined above, if 5 μM CSA in solution results in a10%, or lower, reduction in RUs of dAb binding to HSA, then there isconsidered to be no competition for binding. Accordingly, a reduction inRUs of dAb binding to HSA in the presence of CSA of greater than 10%would indicate the presence of competition for binding of the dAb forbinding HSA by CSA. A reduction in RUs of dAb binding to HSA of lessthan 10% would indicate the absence of competition by CSA for the dAb'sbinding HSA, with reductions of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1%being progressively more stringent requirements for indicating theabsence of competition. The greater the reduction in RUs of dAb bindingto HSA, the greater the competition. Thus, increasing levels ofcompetition can be graded according to the percent reduction in RUsbinding to HSA, i.e. at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to 100% reduction.

A fragment as used herein refers to less than 100% of the sequence(e.g., up to 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), butcomprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or more contiguous amino acids. A fragment is ofsufficient length such that the the serum albumin binding of interest ismaintained with affinity of 1×10⁻⁶ M or less. A fragment as used hereinalso refers to optional insertions, deletions and substitutions of oneor more amino acids which do not substantially alter the ability of thealtered polypeptide to bind to a single domain antibody raised againstthe target. The number of amino acid insertions deletions orsubstitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69 or 70 amino acids.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridisation techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc. which are incorporated herein by reference)and chemical methods. Standards techniques for surface plasmon resonanceassays include Jan Terje Andersen et al. (2006) Eur. J. Immunol.36:304-3051 Fagerstam (1991) Tech. Protein Chem. 2:65-71, and Johnssonet al (1991) Anal. Biochem 198:268-277.

Preparation of Immunoglobulin Based Multi-Specific Ligands

Dual specific ligands according to the invention, whether open or closedin conformation according to the desired configuration of the invention,may be prepared according to previously established techniques, used inthe field of antibody engineering, for the preparation of scFv, “phage”antibodies and other engineered antibody molecules. Techniques for thepreparation of antibodies, and in particular bispecific antibodies, arefor example described in the following reviews and the references citedtherein: Winter & Milstein, (1991) Nature 349:293-299; Plueckthun (1992)Immunological Reviews 130:151-188; Wright et al., (1992) Crit. Rev.Immunol. 12:125-168; Holliger, P. & Winter, G. (1993) Curr. Op.Biotechn. 4, 446-449; Carter, et al. (1995) J. Hematother. 4, 463-470;Chester, K. A. & Hawkins, R. E. (1995) Trends Biotechn. 13, 294-300;Hoogenboom, H. R. (1997) Nature Biotechnol. 15, 125-126; Fearon, D.(1997) Nature Biotechnol. 15, 618-619; Plückthun, A. & Pack, P. (1997)Immunotechnology 3, 83-105; Carter, P. & Merchant, A. M. (1997) Curr.Opin. Biotechnol. 8, 449-454; Holliger, P. & Winter, G. (1997) CancerImmunol. Immunother. 45, 128-130.

The invention provides for the selection of variable domains against twodifferent antigens or epitopes, and subsequent combination of thevariable domains.

The techniques employed for selection of the variable domains employlibraries and selection procedures which are known in the art. Naturallibraries (Marks et al. (1991) J. Mol. Biol., 222: 581; Vaughan et al.(1996) Nature Biotech., 14: 309) which use rearranged V genes harvestedfrom human B cells are well known to those skilled in the art. Syntheticlibraries (Hoogenboom & Winter (1992) J. Mol. Biol., 227: 381; Barbas etal. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al. (1994)EMBO J., 13: 692; Griffiths et al. (1994) EMBO J., 13: 3245; De Kruif etal. (1995) J Mol. Biol., 248: 97) are prepared by cloning immunoglobulinV genes, usually using PCR. Errors in the PCR process can lead to a highdegree of randomisation. V_(H) and/or V_(L) libraries may be selectedagainst target antigens or epitopes separately, in which case singledomain binding is directly selected for, or together.

A preferred method for making a dual specific ligand according to thepresent invention comprises using a selection system in which arepertoire of variable domains is selected for binding to a firstantigen or epitope and a repertoire of variable domains is selected forbinding to a second antigen or epitope. The selected variable first andsecond variable domains are then combined and the dual-specific ligandselected for binding to both first and second antigen or epitope. Closedconformation ligands are selected for binding both first and secondantigen or epitope in isolation but not simultaneously.

A. Library Vector Systems

A variety of selection systems are known in the art which are suitablefor use in the present invention. Examples of such systems are describedbelow.

Bacteriophage lambda expression systems may be screened directly asbacteriophage plaques or as colonies of lysogens, both as previouslydescribed (Huse et al. (1989) Science, 246: 1275; Caton and Koprowski(1990) Proc. Natl. Acad. Sci. U.S.A., 87; Mullinax et al. (1990) Proc.Natl. Acad. Sci. USA., 87: 8095; Persson et al. (1991) Proc. Natl. Acad.Sci. USA., 88: 2432) and are of use in the invention. Whilst suchexpression systems can be used to screen up to 10⁶ different members ofa library, they are not really suited to screening of larger numbers(greater than 10⁶ members).

Of particular use in the construction of libraries are selection displaysystems, which enable a nucleic acid to be linked to the polypeptide itexpresses. As used herein, a selection display system is a system thatpermits the selection, by suitable display means, of the individualmembers of the library by binding the generic and/or target ligands.

Selection protocols for isolating desired members of large libraries areknown in the art, as typified by phage display techniques. Such systems,in which diverse peptide sequences are displayed on the surface offilamentous bacteriophage (Scott and Smith (1990) Science, 249: 386),have proven useful for creating libraries of antibody fragments (and thenucleotide sequences that encoding them) for the in vitro selection andamplification of specific antibody fragments that bind a target antigen(McCafferty et al., WO 92/01047). The nucleotide sequences encoding theV_(H) and V_(L) regions are linked to gene fragments which encode leadersignals that direct them to the periplasmic space of E. coli and as aresult the resultant antibody fragments are displayed on the surface ofthe bacteriophage, typically as fusions to bacteriophage coat proteins(e.g., pIII or pVIII). Alternatively, antibody fragments are displayedexternally on lambda phage capsids (phagebodies). An advantage ofphage-based display systems is that, because they are biologicalsystems, selected library members can be amplified simply by growing thephage containing the selected library member in bacterial cells.Furthermore, since the nucleotide sequence that encode the polypeptidelibrary member is contained on a phage or phagemid vector, sequencing,expression and subsequent genetic manipulation is relativelystraightforward.

Methods for the construction of bacteriophage antibody display librariesand lambda phage expression libraries are well known in the art(McCafferty et al. (1990) Nature, 348: 552; Kang et al. (1991) Proc.Natl. Acad. Sci. USA., 88: 4363; Clackson et al. (1991) Nature, 352:624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton et al. (1991)Proc. Natl. Acad. Sci USA., 88: 10134; Hoogenboom et al. (1991) NucleicAcids Res., 19: 4133; Chang et al. (1991) J. Immunol., 147: 3610;Breitling et al. (1991) Gene, 104: 147; Marks et al. (1991) supra;Barbas et al. (1992) supra; Hawkins and Winter (1992) J. Immunol., 22:867; Marks et al., 1992, J. Biol. Chem., 267: 16007; Lerner et al.(1992) Science, 258: 1313, incorporated herein by reference).

One particularly advantageous approach has been the use of scFvphage-libraries (Huston et al., 1988, Proc. Natl. Acad. Sci U.S.A., 85:5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad. Sci U.S.A., 87:1066-1070; McCafferty et al. (1990) supra; Clackson et al. (1991)Nature, 352: 624; Marks et al. (1991) J. Mol. Biol., 222: 581; Chiswellet al. (1992) Trends Biotech., 10: 80; Marks et al. (1992) J. Biol.Chem., 267). Various embodiments of scFv libraries displayed onbacteriophage coat proteins have been described. Refinements of phagedisplay approaches are also known, for example as described inWO96/06213 and WO92/01047 (Medical Research Council et al.) andWO97/08320 (Morphosys), which are incorporated herein by reference.

Other systems for generating libraries of polypeptides involve the useof cell-free enzymatic machinery for the in vitro synthesis of thelibrary members. In one method, RNA molecules are selected by alternaterounds of selection against a target ligand and PCR amplification (Tuerkand Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature,346: 818). A similar technique may be used to identify DNA sequenceswhich bind a predetermined human transcription factor (Thiesen and Bach(1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science,257: 635; WO92/05258 and WO92/14843). In a similar way, in vitrotranslation can be used to synthesise polypeptides as a method forgenerating large libraries. These methods which generally comprisestabilised polysome complexes, are described further in WO88/08453,WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536.Alternative display systems which are not phage-based, such as thosedisclosed in WO95/22625 and WO95/11922 (Affymax) use the polysomes todisplay polypeptides for selection.

A still further category of techniques involves the selection ofrepertoires in artificial compartments, which allow the linkage of agene with its gene product. For example, a selection system in whichnucleic acids encoding desirable gene products may be selected inmicrocapsules formed by water-in-oil emulsions is described inWO99/02671, WO00/40712 and Tawfik & Griffiths (1998) Nature Biotechnol16(7), 652-6. Genetic elements encoding a gene product having a desiredactivity are compartmentalised into microcapsules and then transcribedand/or translated to produce their respective gene products (RNA orprotein) within the microcapsules. Genetic elements which produce geneproduct having desired activity are subsequently sorted. This approachselects gene products of interest by detecting the desired activity by avariety of means.

B. Library Construction.

Libraries intended for selection, may be constructed using techniquesknown in the art, for example as set forth above, or may be purchasedfrom commercial sources. Libraries which are useful in the presentinvention are described, for example, in WO99/20749. Once a vectorsystem is chosen and one or more nucleic acid sequences encodingpolypeptides of interest are cloned into the library vector, one maygenerate diversity within the cloned molecules by undertakingmutagenesis prior to expression; alternatively, the encoded proteins maybe expressed and selected, as described above, before mutagenesis andadditional rounds of selection are performed. Mutagenesis of nucleicacid sequences encoding structurally optimised polypeptides is carriedout by standard molecular methods. Of particular use is the polymerasechain reaction, or PCR, (Mullis and Faloona (1987) Methods Enzymol.,155: 335, herein incorporated by reference). PCR, which uses multiplecycles of DNA replication catalysed by a thermostable, DNA-dependent DNApolymerase to amplify the target sequence of interest, is well known inthe art. The construction of various antibody libraries has beendiscussed in Winter et al. (1994) Ann. Rev. Immunology 12, 433-55, andreferences cited therein.

PCR is performed using template DNA (at least lfg; more usefully, 1-1000ng) and at least 25 pmol of oligonucleotide primers; it may beadvantageous to use a larger amount of primer when the primer pool isheavily heterogeneous, as each sequence is represented by only a smallfraction of the molecules of the pool, and amounts become limiting inthe later amplification cycles. A typical reaction mixture includes: 2μl of DNA, 25 pmol of oligonucleotide primer, 2.5 μl of 10×PCR buffer 1(Perkin-Elmer, Foster City, Calif.), 0.4 μl of 1.25 μM dNTP, 0.15 μl (or2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, Calif.) anddeionized water to a total volume of 25 μl. Mineral oil is overlaid andthe PCR is performed using a programmable thermal cycler. The length andtemperature of each step of a PCR cycle, as well as the number ofcycles, is adjusted in accordance to the stringency requirements ineffect. Annealing temperature and timing are determined both by theefficiency with which a primer is expected to anneal to a template andthe degree of mismatch that is to be tolerated; obviously, when nucleicacid molecules are simultaneously amplified and mutagenised, mismatch isrequired, at least in the first round of synthesis. The ability tooptimise the stringency of primer annealing conditions is well withinthe knowledge of one of moderate skill in the art. An annealingtemperature of between 30° C. and 72° C. is used. Initial denaturationof the template molecules normally occurs at between 92° C. and 99° C.for 4 minutes, followed by 20-40 cycles consisting of denaturation(94-99° C. for 15 seconds to 1 minute), annealing (temperaturedetermined as discussed above; 1-2 minutes), and extension (72° C. for1-5 minutes, depending on the length of the amplified product). Finalextension is generally for 4 minutes at 72° C., and may be followed byan indefinite (0-24 hour) step at 4° C.

C. Combining Single Variable Domains

Domains useful in the invention, once selected, may be combined by avariety of methods known in the art, including covalent and non-covalentmethods.

Preferred methods include the use of polypeptide linkers, as described,for example, in connection with scFv molecules (Bird et al., (1988)Science 242:423-426). Discussion of suitable linkers is provided in Birdet al. Science 242, 423-426; Hudson et al, Journal Immunol Methods 231(1999) 177-189; Hudson et al, Proc Nat Acad Sci USA 85, 5879-5883.Linkers are preferably flexible, allowing the two single domains tointeract. One linker example is a (Glyn Ser)_(n) linker, where n=1 to 8,e.g., 2, 3, 4, 5 or 7. The linkers used in diabodies, which are lessflexible, may also be employed (Holliger et al., (1993) PNAS (USA)90:6444-6448).

In one embodiment, the linker employed is not an immunoglobulin hingeregion.

Variable domains may be combined using methods other than linkers. Forexample, the use of disulphide bridges, provided throughnaturally-occurring or engineered cysteine residues, may be exploited tostabilise V_(H)-V_(H), V_(L)-V_(L) or V_(H)-V_(L) dimers (Reiter et al.,(1994) Protein Eng. 7:697-704) or by remodelling the interface betweenthe variable domains to improve the “fit” and thus the stability ofinteraction (Ridgeway et al., (1996) Protein Eng. 7:617-621; Zhu et al.,(1997) Protein Science 6:781-788).

Other techniques for joining or stabilising variable domains ofimmunoglobulins, and in particular antibody V_(H) domains, may beemployed as appropriate.

In accordance with the present invention, dual specific ligands can bein “closed” conformations in solution. A “closed” configuration is thatin which the two domains (for example V_(H) and V_(L)) are present inassociated form, such as that of an associated V_(H)-V_(L) pair whichforms an antibody binding site. For example, scFv may be in a closedconformation, depending on the arrangement of the linker used to linkthe V_(H) and V_(L) domains. If this is sufficiently flexible to allowthe domains to associate, or rigidly holds them in the associatedposition, it is likely that the domains will adopt a closedconformation.

Similarly, V_(H) domain pairs and V_(L) domain pairs may exist in aclosed conformation. Generally, this will be a function of closeassociation of the domains, such as by a rigid linker, in the ligandmolecule. Ligands in a closed conformation will be unable to bind boththe molecule which increases the half-life of the ligand and a secondtarget molecule. Thus, the ligand will typically only bind the secondtarget molecule on dissociation from the molecule which increases thehalf-life of the ligand.

Moreover, the construction of V_(H)/V_(H), V_(L)/V_(L) or V_(H)/V_(L)dimers without linkers provides for competition between the domains.

Ligands according to the invention may moreover be in an openconformation. In such a conformation, the ligands will be able tosimultaneously bind both the molecule which increases the half-life ofthe ligand and the second target molecule. Typically, variable domainsin an open configuration are (in the case of V_(H)-V_(L) pairs) held farenough apart for the domains not to interact and form an antibodybinding site and not to compete for binding to their respectiveepitopes. In the case of V_(H)/V_(H) or V_(L)/V_(L) dimers, the domainsare not forced together by rigid linkers. Naturally, such domainpairings will not compete for antigen binding or form an antibodybinding site.

Fab fragments and whole antibodies will exist primarily in the closedconformation, although it will be appreciated that open and closed dualspecific ligands are likely to exist in a variety of equilibria underdifferent circumstances. Binding of the ligand to a target is likely toshift the balance of the equilibrium towards the open configuration.Thus, certain ligands according to the invention can exist in twoconformations in solution, one of which (the open form) can bind twoantigens or epitopes independently, whilst the alternative conformation(the closed form) can only bind one antigen or epitope; antigens orepitopes thus compete for binding to the ligand in this conformation.

Although the open form of the dual specific ligand may thus exist inequilibrium with the closed form in solution, it is envisaged that theequilibrium will favour the closed form; moreover, the open form can besequestered by target binding into a closed conformation. Preferably,therefore, certain dual specific ligands of the invention are present inan equilibrium between two (open and closed) conformations.

Dual specific ligands according to the invention may be modified inorder to favour an open or closed conformation. For example,stabilisation of V_(H)-V_(L) interactions with disulphide bondsstabilises the closed conformation. Moreover, linkers used to join thedomains, including V_(H) domain and V_(L) domain pairs, may beconstructed such that the open from is favoured; for example, thelinkers may sterically hinder the association of the domains, such as byincorporation of large amino acid residues in opportune locations, orthe designing of a suitable rigid structure which will keep the domainsphysically spaced apart.

D. Characterisation of the Dual-Specific Ligand.

The binding of the dual-specific ligand to its specific antigens orepitopes can be tested by methods which will be familiar to thoseskilled in the art and include ELISA. In a preferred embodiment of theinvention binding is tested using monoclonal phage ELISA.

Phage ELISA may be performed according to any suitable procedure: anexemplary protocol is set forth below.

Populations of phage produced at each round of selection can be screenedfor binding by ELISA to the selected antigen or epitope, to identify“polyclonal” phage antibodies. Phage from single infected bacterialcolonies from these populations can then be screened by ELISA toidentify “monoclonal” phage antibodies. It is also desirable to screensoluble antibody fragments for binding to antigen or epitope, and thiscan also be undertaken by ELISA using reagents, for example, against aC- or N-terminal tag (see for example Winter et al. (1994) Ann. Rev.Immunology 12, 433-55 and references cited therein.

The diversity of the selected phage monoclonal antibodies may also beassessed by gel electrophoresis of PCR products (Marks et al. 1991,supra; Nissim et al. 1994 supra), probing (Tomlinson et al., 1992) J.Mol. Biol. 227, 776) or by sequencing of the vector DNA.

E. Structure of ‘Dual-Specific Ligands’.

As described above, an antibody is herein defined as an antibody (forexample IgG, IgM, IgA, IgA, IgE) or fragment (Fab, Fv, disulphide linkedFv, scFv, diabody) which comprises at least one heavy and a light chainvariable domain, at least two heavy chain variable domains or at leasttwo light chain variable domains. It may be at least partly derived fromany species naturally producing an antibody, or created by recombinantDNA technology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria).

In a preferred embodiment of the invention the dual-specific ligandcomprises at least one single heavy chain variable domain of an antibodyand one single light chain variable domain of an antibody, or two singleheavy or light chain variable domains. For example, the ligand maycomprise a V_(H)/V_(L) pair, a pair of V_(H) domains or a pair of V_(L)domains.

The first and the second variable domains of such a ligand may be on thesame polypeptide chain. Alternatively they may be on separatepolypeptide chains. In the case that they are on the same polypeptidechain they may be linked by a linker, which is preferentially a peptidesequence, as described above.

The first and second variable domains may be covalently ornon-covalently associated. In the case that they are covalentlyassociated, the covalent bonds may be disulphide bonds.

In the case that the variable domains are selected from V-generepertoires selected for instance using phage display technology asherein described, then these variable domains comprise a universalframework region, such that is they may be recognised by a specificgeneric ligand as herein defined. The use of universal frameworks,generic ligands and the like is described in WO99/20749.

Where V-gene repertoires are used variation in polypeptide sequence ispreferably located within the structural loops of the variable domains.The polypeptide sequences of either variable domain may be altered byDNA shuffling or by mutation in order to enhance the interaction of eachvariable domain with its complementary pair. DNA shuffling is known inthe art and taught, for example, by Stemmer, 1994, Nature 370: 389-391and U.S. Pat. No. 6,297,053, both of which are incorporated herein byreference. Other methods of mutagenesis are well known to those of skillin the art.

In a preferred embodiment of the invention the ‘dual-specific ligand’ isa single chain Fv fragment. In an alternative embodiment of theinvention, the ‘dual-specific ligand’ consists of a Fab format.

In a further aspect, the present invention provides nucleic acidencoding at least a ‘dual-specific ligand’ as herein defined.

One skilled in the art will appreciate that, depending on the aspect ofthe invention, both antigens or epitopes may bind simultaneously to thesame antibody molecule. Alternatively, they may compete for binding tothe same antibody molecule. For example, where both epitopes are boundsimultaneously, both variable domains of a dual specific ligand are ableto independently bind their target epitopes. Where the domains compete,the one variable domain is capable of binding its target, but not at thesame time as the other variable domain binds its cognate target; or thefirst variable domain is capable of binding its target, but not at thesame time as the second variable domain binds its cognate target.

The variable domains may be derived from antibodies directed againsttarget antigens or epitopes. Alternatively they may be derived from arepertoire of single antibody domains such as those expressed on thesurface of filamentous bacteriophage. Selection may be performed asdescribed below.

In general, the nucleic acid molecules and vector constructs requiredfor the performance of the present invention may be constructed andmanipulated as set forth in standard laboratory manuals, such asSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, USA.

The manipulation of nucleic acids useful in the present invention istypically carried out in recombinant vectors.

Thus in a further aspect, the present invention provides a vectorcomprising nucleic acid encoding at least a ‘dual-specific ligand’ asherein defined.

As used herein, vector refers to a discrete element that is used tointroduce heterologous DNA into cells for the expression and/orreplication thereof. Methods by which to select or construct and,subsequently, use such vectors are well known to one of ordinary skillin the art. Numerous vectors are publicly available, including bacterialplasmids, bacteriophage, artificial chromosomes and episomal vectors.Such vectors may be used for simple cloning and mutagenesis;alternatively gene expression vector is employed. A vector of useaccording to the invention may be selected to accommodate a polypeptidecoding sequence of a desired size, typically from 0.25 kilobase (kb) to40 kb or more in length. A suitable host cell is transformed with thevector after in vitro cloning manipulations. Each vector containsvarious functional components, which generally include a cloning (or“polylinker”) site, an origin of replication and at least one selectablemarker gene. If given vector is an expression vector, it additionallypossesses one or more of the following: enhancer element, promoter,transcription termination and signal sequences, each positioned in thevicinity of the cloning site, such that they are operatively linked tothe gene encoding a ligand according to the invention.

Both cloning and expression vectors generally contain nucleic acidsequences that enable the vector to replicate in one or more selectedhost cells. Typically in cloning vectors, this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2 micron plasmid origin issuitable for yeast, and various viral origins (e.g. SV 40, adenovirus)are useful for cloning vectors in mammalian cells. Generally, the originof replication is not needed for mammalian expression vectors unlessthese are used in mammalian cells able to replicate high levels of DNA,such as COS cells.

Advantageously, a cloning or expression vector may contain a selectiongene also referred to as selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Host cells not transformed with the vectorcontaining the selection gene will therefore not survive in the culturemedium. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexateor tetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available in the growth media.

Since the replication of vectors encoding a ligand according to thepresent invention is most conveniently performed in E. coli, an E.coli-selectable marker, for example, the β-lactamase gene that confersresistance to the antibiotic ampicillin, is of use. These can beobtained from E. coli plasmids, such as pBR322 or a pUC plasmid such aspUC18 or pUC19.

Expression vectors usually contain a promoter that is recognized by thehost organism and is operably linked to the coding sequence of interest.Such a promoter may be inducible or constitutive. The term “operablylinked” refers to a juxtaposition wherein the components described arein a relationship permitting them to function in their intended manner.A control sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved underconditions compatible with the control sequences.

Promoters suitable for use with prokaryotic hosts include, for example,the β-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system and hybrid promoters such as the tacpromoter. Promoters for use in bacterial systems will also generallycontain a Shine-Delgarno sequence operably linked to the codingsequence.

The preferred vectors are expression vectors that enables the expressionof a nucleotide sequence corresponding to a polypeptide library member.Thus, selection with the first and/or second antigen or epitope can beperformed by separate propagation and expression of a single cloneexpressing the polypeptide library member or by use of any selectiondisplay system. As described above, the preferred selection displaysystem is bacteriophage display. Thus, phage or phagemid vectors may beused, e.g. pIT1 or pIT2. Leader sequences useful in the inventioninclude pelB, stII, ompA, phoA, bla and pelA. One example are phagemidvectors which have an E. coli. origin of replication (for doublestranded replication) and also a phage origin of replication (forproduction of single-stranded DNA). The manipulation and expression ofsuch vectors is well known in the art (Hoogenboom and Winter (1992)supra; Nissim et al. (1994) supra). Briefly, the vector contains aβ-lactamase gene to confer selectivity on the phagemid and a lacpromoter upstream of a expression cassette that consists (N to Cterminal) of a pelB leader sequence (which directs the expressedpolypeptide to the periplasmic space), a multiple cloning site (forcloning the nucleotide version of the library member), optionally, oneor more peptide tag (for detection), optionally, one or more TAG stopcodon and the phage protein pIII. Thus, using various suppressor andnon-suppressor strains of E. coli and with the addition of glucose,iso-propyl thio-β-D-galactoside (IPTG) or a helper phage, such as VCSM13, the vector is able to replicate as a plasmid with no expression,produce large quantities of the polypeptide library member only orproduce phage, some of which contain at least one copy of thepolypeptide-pIII fusion on their surface.

Construction of vectors encoding ligands according to the inventionemploys conventional ligation techniques. Isolated vectors or DNAfragments are cleaved, tailored, and religated in the form desired togenerate the required vector. If desired, analysis to confirm that thecorrect sequences are present in the constructed vector can be performedin a known fashion. Suitable methods for constructing expressionvectors, preparing in vitro transcripts, introducing DNA into hostcells, and performing analyses for assessing expression and function areknown to those skilled in the art. The presence of a gene sequence in asample is detected, or its amplification and/or expression quantified byconventional methods, such as Southern or Northern analysis, Westernblotting, dot blotting of DNA, RNA or protein, in situ hybridisation,immunocytochemistry or sequence analysis of nucleic acid or proteinmolecules. Those skilled in the art will readily envisage how thesemethods may be modified, if desired.

Structure of Closed Conformation Multispecific Ligands

According to one aspect of the second configuration of the inventionpresent invention, the two or more non-complementary epitope bindingdomains are linked so that they are in a closed conformation as hereindefined. Advantageously, they may be further attached to a skeletonwhich may, as an alternative, or in addition to a linker describedherein, facilitate the formation and/or maintenance of the closedconformation of the epitope binding sites with respect to one another.

(I) Skeletons

Skeletons may be based on immunoglobulin molecules or may benon-immunoglobulin in origin as set forth above. Preferredimmunoglobulin skeletons as herein defined includes any one or more ofthose selected from the following: an immunoglobulin molecule comprisingat least (i) the CL (kappa or lambda subclass) domain of an antibody; or(ii) the CH1 domain of an antibody heavy chain; an immunoglobulinmolecule comprising the CH1 and CH2 domains of an antibody heavy chain;an immunoglobulin molecule comprising the CH1, CH2 and CH3 domains of anantibody heavy chain; or any of the subset (ii) in conjunction with theCL (kappa or lambda subclass) domain of an antibody. A hinge regiondomain may also be included. Such combinations of domains may, forexample, mimic natural antibodies, such as IgG or IgM, or fragmentsthereof, such as Fv, scFv, Fab or F(ab′)₂ molecules. Those skilled inthe art will be aware that this list is not intended to be exhaustive.

(II) Protein Scaffolds

Each epitope binding domain comprises a protein scaffold and one or moreCDRs which are involved in the specific interaction of the domain withone or more epitopes. Advantageously, an epitope binding domainaccording to the present invention comprises three CDRs. Suitableprotein scaffolds include any of those selected from the groupconsisting of the following: those based on immunoglobulin domains,those based on fibronectin, those based on affibodies, those based onCTLA4, those based on chaperones such as GroEL, those based onlipocallin and those based on the bacterial Fc receptors SpA, and SpD.Those skilled in the art will appreciate that this list is not intendedto be exhaustive.

F: Scaffolds for Use in Constructing Dual Specific Ligands

i. Selection of the Main-Chain Conformation

The members of the immunoglobulin superfamily all share a similar foldfor their polypeptide chain. For example, although antibodies are highlydiverse in terms of their primary sequence, comparison of sequences andcrystallographic structures has revealed that, contrary to expectation,five of the six antigen binding loops of antibodies (H1, H2, L1, L2, L3)adopt a limited number of main-chain conformations, or canonicalstructures (Chothia and Lesk (1987) J. Mol. Biol., 196: 901; Chothia etal. (1989) Nature, 342: 877). Analysis of loop lengths and key residueshas therefore enabled prediction of the main-chain conformations of H1,H2, L1, L2 and L3 found in the majority of human antibodies (Chothia etal. (1992) J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J., 14:4628; Williams et al. (1996) J. Mol. Biol., 264: 220). Although the H3region is much more diverse in terms of sequence, length and structure(due to the use of D segments), it also forms a limited number ofmain-chain conformations for short loop lengths which depend on thelength and the presence of particular residues, or types of residue, atkey positions in the loop and the antibody framework (Martin et al.(1996) J. Mol. Biol., 263: 800; Shirai et al. (1996) FEBS Letters, 399:1).

The dual specific ligands of the present invention are advantageouslyassembled from libraries of domains, such as libraries of V_(H) domainsand/or libraries of V_(L) domains. Moreover, the dual specific ligandsof the invention may themselves be provided in the form of libraries. Inone aspect of the present invention, libraries of dual specific ligandsand/or domains are designed in which certain loop lengths and keyresidues have been chosen to ensure that the main-chain conformation ofthe members is known. Advantageously, these are real conformations ofimmunoglobulin superfamily molecules found in nature, to minimise thechances that they are non-functional, as discussed above. Germ line Vgene segments serve as one suitable basic framework for constructingantibody or T-cell receptor libraries; other sequences are also of use.Variations may occur at a low frequency, such that a small number offunctional members may possess an altered main-chain conformation, whichdoes not affect its function.

Canonical structure theory is also of use to assess the number ofdifferent main-chain conformations encoded by ligands, to predict themain-chain conformation based on ligand sequences and to chose residuesfor diversification which do not affect the canonical structure. It isknown that, in the human V_(κ) domain, the L1 loop can adopt one of fourcanonical structures, the L2 loop has a single canonical structure andthat 90% of human V_(κ) domains adopt one of four or five canonicalstructures for the L3 loop (Tomlinson et al. (1995) supra); thus, in theV_(κ) domain alone, different canonical structures can combine to createa range of different main-chain conformations. Given that the V_(λ)domain encodes a different range of canonical structures for the L1, L2and L3 loops and that V_(κ) and V_(λ) domains can pair with any V_(H)domain which can encode several canonical structures for the H1 and H2loops, the number of canonical structure combinations observed for thesefive loops is very large. This implies that the generation of diversityin the main-chain conformation may be essential for the production of awide range of binding specificities. However, by constructing anantibody library based on a single known main-chain conformation it hasbeen found, contrary to expectation, that diversity in the main-chainconformation is not required to generate sufficient diversity to targetsubstantially all antigens. Even more surprisingly, the singlemain-chain conformation need not be a consensus structure—a singlenaturally occurring conformation can be used as the basis for an entirelibrary. Thus, in a preferred aspect, the dual-specific ligands of theinvention possess a single known main-chain conformation.

The single main-chain conformation that is chosen is preferablycommonplace among molecules of the immunoglobulin superfamily type inquestion. A conformation is commonplace when a significant number ofnaturally occurring molecules are observed to adopt it. Accordingly, ina preferred aspect of the invention, the natural occurrence of thedifferent main-chain conformations for each binding loop of animmunoglobulin domain are considered separately, and then a naturallyoccurring variable domain is chosen which possesses the desiredcombination of main-chain conformations for the different loops. If noneis available, the nearest equivalent may be chosen. It is preferablethat the desired combination of main-chain conformations for thedifferent loops is created by selecting germ line gene segments whichencode the desired main-chain conformations. It is more preferable, thatthe selected germ line gene segments are frequently expressed in nature,and most preferable that they are the most frequently expressed of allnatural germ line gene segments.

In designing dual specific ligands or libraries thereof the incidence ofthe different main-chain conformations for each of the six antigenbinding loops may be considered separately. For H1, H2, L1, L2 and L3, agiven conformation that is adopted by between 20% and 100% of theantigen binding loops of naturally occurring molecules is chosen.Typically, its observed incidence is above 35% (i.e. between 35% and100%) and, ideally, above 50% or even above 65%. Since the vast majorityof H3 loops do not have canonical structures, it is preferable to selecta main-chain conformation which is commonplace among those loops whichdo display canonical structures. For each of the loops, the conformationwhich is observed most often in the natural repertoire is thereforeselected. In human antibodies, the most popular canonical structures(CS) for each loop are as follows: H1—CS 1 (79% of the expressedrepertoire), H2—CS 3 (46%), L1—CS 2 of V_(κ) (39%), L2—CS 1 (100%),L3—CS 1 of V_(κ) (36%) (calculation assumes a κ:λ ratio of 70:30, Hoodet al. (1967) Cold Spring Harbor Symp. Quant. Biol., 48: 133). For H3loops that have canonical structures, a CDR3 length (Kabat et al. (1991)Sequences of proteins of immunological interest, U.S. Department ofHealth and Human Services) of seven residues with a salt-bridge fromresidue 94 to residue 101 appears to be the most common. There are atleast 16 human antibody sequences in the EMBL data library with therequired H3 length and key residues to form this conformation and atleast two crystallographic structures in the protein data bank which canbe used as a basis for antibody modelling (2cgr and ltet). The mostfrequently expressed germ line gene segments that this combination ofcanonical structures are the V_(H) segment 3-23 (DP-47), the J_(H)segment JH4b, the V_(κ) segment O2/O12 (DPK9) and the J_(κ) segmentJ_(κ)1. V_(H) segments DP45 and DP38 are also suitable. These segmentscan therefore be used in combination as a basis to construct a librarywith the desired single main-chain conformation.

Alternatively, instead of choosing the single main-chain conformationbased on the natural occurrence of the different main-chainconformations for each of the binding loops in isolation, the naturaloccurrence of combinations of main-chain conformations is used as thebasis for choosing the single main-chain conformation. In the case ofantibodies, for example, the natural occurrence of canonical structurecombinations for any two, three, four, five or for all six of theantigen binding loops can be determined. Here, it is preferable that thechosen conformation is commonplace in naturally occurring antibodies andmost preferable that it observed most frequently in the naturalrepertoire. Thus, in human antibodies, for example, when naturalcombinations of the five antigen binding loops, H1, H2, L1, L2 and L3,are considered, the most frequent combination of canonical structures isdetermined and then combined with the most popular conformation for theH3 loop, as a basis for choosing the single main-chain conformation.

ii. Diversification of the Canonical Sequence

Having selected several known main-chain conformations or, preferably asingle known main-chain conformation, dual specific ligands according tothe invention or libraries for use in the invention can be constructedby varying the binding site of the molecule in order to generate arepertoire with structural and/or functional diversity. This means thatvariants are generated such that they possess sufficient diversity intheir structure and/or in their function so that they are capable ofproviding a range of activities.

The desired diversity is typically generated by varying the selectedmolecule at one or more positions. The positions to be changed can bechosen at random or are preferably selected. The variation can then beachieved either by randomization, during which the resident amino acidis replaced by any amino acid or analogue thereof, natural or synthetic,producing a very large number of variants or by replacing the residentamino acid with one or more of a defined subset of amino acids,producing a more limited number of variants.

Various methods have been reported for introducing such diversity.Error-prone PCR (Hawkins et al. (1992) J. Mol. Biol., 226: 889),chemical mutagenesis (Deng et al. (1994) J. Biol. Chem., 269: 9533) orbacterial mutator strains (Low et al. (1996) J. Mol. Biol., 260: 359)can be used to introduce random mutations into the genes that encode themolecule. Methods for mutating selected positions are also well known inthe art and include the use of mismatched oligonucleotides or degenerateoligonucleotides, with or without the use of PCR. For example, severalsynthetic antibody libraries have been created by targeting mutations tothe antigen binding loops. The H3 region of a human tetanustoxoid-binding Fab has been randomised to create a range of new bindingspecificities (Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4457). Random or semi-random H3 and L3 regions have been appended togerm line V gene segments to produce large libraries with unmutatedframework regions (Hoogenboom & Winter (1992) J. Mol. Biol., 227: 381;Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al.(1994) EMBO J, 13: 692; Griffiths et al. (1994) EMBO J., 13: 3245; DeKruif et al. (1995) J. Mol. Biol., 248: 97). Such diversification hasbeen extended to include some or all of the other antigen binding loops(Crameri et al. (1996) Nature Med., 2: 100; Riechmann et al. (1995)Bio/Technology, 13: 475; Morphosys, WO97/08320, supra).

Since loop randomization has the potential to create approximately morethan 10¹⁵ structures for H3 alone and a similarly large number ofvariants for the other five loops, it is not feasible using currenttransformation technology or even by using cell free systems to producea library representing all possible combinations. For example, in one ofthe largest libraries constructed to date, 6×10¹⁰ different antibodies,which is only a fraction of the potential diversity for a library ofthis design, were generated (Griffiths et al. (1994) supra).

In a preferred embodiment, only those residues which are directlyinvolved in creating or modifying the desired function of the moleculeare diversified. For many molecules, the function will be to bind atarget and therefore diversity should be concentrated in the targetbinding site, while avoiding changing residues which are crucial to theoverall packing of the molecule or to maintaining the chosen main-chainconformation.

Diversification of the Canonical Sequence as it Applies to AntibodyDomains

In the case of antibody dual-specific ligands, the binding site for thetarget is most often the antigen binding site. Thus, in a highlypreferred aspect, the invention provides libraries of or for theassembly of antibody dual-specific ligands in which only those residuesin the antigen binding site are varied. These residues are extremelydiverse in the human antibody repertoire and are known to make contactsin high-resolution antibody/antigen complexes. For example, in L2 it isknown that positions 50 and 53 are diverse in naturally occurringantibodies and are observed to make contact with the antigen. Incontrast, the conventional approach would have been to diversify all theresidues in the corresponding Complementarity Determining Region (CDR1)as defined by Kabat et al. (1991, supra), some seven residues comparedto the two diversified in the library for use according to theinvention. This represents a significant improvement in terms of thefunctional diversity required to create a range of antigen bindingspecificities.

In nature, antibody diversity is the result of two processes: somaticrecombination of germ line V, D and J gene segments to create a naiveprimary repertoire (so called germ line and junctional diversity) andsomatic hypermutation of the resulting rearranged V genes. Analysis ofhuman antibody sequences has shown that diversity in the primaryrepertoire is focused at the centre of the antigen binding site whereassomatic hypermutation spreads diversity to regions at the periphery ofthe antigen binding site that are highly conserved in the primaryrepertoire (see Tomlinson et al. (1996) J. Mol. Biol., 256: 813). Thiscomplementarity has probably evolved as an efficient strategy forsearching sequence space and, although apparently unique to antibodies,it can easily be applied to other polypeptide repertoires. The residueswhich are varied are a subset of those that form the binding site forthe target. Different (including overlapping) subsets of residues in thetarget binding site are diversified at different stages duringselection, if desired.

In the case of an antibody repertoire, an initial ‘naive’ repertoire iscreated where some, but not all, of the residues in the antigen bindingsite are diversified. As used herein in this context, the term “naive”refers to antibody molecules that have no pre-determined target. Thesemolecules resemble those which are encoded by the immunoglobulin genesof an individual who has not undergone immune diversification, as is thecase with fetal and newborn individuals, whose immune systems have notyet been challenged by a wide variety of antigenic stimuli. Thisrepertoire is then selected against a range of antigens or epitopes. Ifrequired, further diversity can then be introduced outside the regiondiversified in the initial repertoire. This matured repertoire can beselected for modified function, specificity or affinity.

The invention provides two different naive repertoires of bindingdomains for the construction of dual specific ligands, or a naivelibrary of dual specific ligands, in which some or all of the residuesin the antigen binding site are varied. The “primary” library mimics thenatural primary repertoire, with diversity restricted to residues at thecenter of the antigen binding site that are diverse in the germ line Vgene segments (germ line diversity) or diversified during therecombination process (junctional diversity). Those residues which arediversified include, but are preferably not limited to, H50, H52, H52a,H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 andL96. In the “somatic” library, diversity is restricted to residues thatare diversified during the recombination process (junctional diversity)or are highly somatically mutated). Those residues which are diversifiedinclude, but are preferably not limited to: H31, H33, H35, H95, H96,H97, H98, L30, L31, L32, L34 and L96. All the residues listed above assuitable for diversification in these libraries are known to makecontacts in one or more antibody-antigen complexes. Since in bothlibraries, not all of the residues in the antigen binding site arevaried, additional diversity is incorporated during selection by varyingthe remaining residues, if it is desired to do so. It shall be apparentto one skilled in the art that any subset of any of these residues (oradditional residues which comprise the antigen binding site) can be usedfor the initial and/or subsequent diversification of the antigen bindingsite.

In the construction of libraries for use in the invention,diversification of chosen positions is typically achieved at the nucleicacid level, by altering the coding sequence which specifies the sequenceof the polypeptide such that a number of possible amino acids (all 20 ora subset thereof) can be incorporated at that position. Using the IUPACnomenclature, the most versatile codon is NNK, which encodes all aminoacids as well as the TAG stop codon. The NNK codon is preferably used inorder to introduce the required diversity. Other codons which achievethe same ends are also of use, including the NNN codon, which leads tothe production of the additional stop codons TGA and TAA.

A feature of side-chain diversity in the antigen binding site of humanantibodies is a pronounced bias which favours certain amino acidresidues. If the amino acid composition of the ten most diversepositions in each of the V_(H), V_(κ) and V_(λ) regions are summed, morethan 76% of the side-chain diversity comes from only seven differentresidues, these being, serine (24%), tyrosine (14%), asparagine (11%),glycine (9%), alanine (7%), aspartate (6%) and threonine (6%). This biastowards hydrophilic residues and small residues which can providemain-chain flexibility probably reflects the evolution of surfaces whichare predisposed to binding a wide range of antigens or epitopes and mayhelp to explain the required promiscuity of antibodies in the primaryrepertoire.

Since it is preferable to mimic this distribution of amino acids, thedistribution of amino acids at the positions to be varied preferablymimics that seen in the antigen binding site of antibodies. Such bias inthe substitution of amino acids that permits selection of certainpolypeptides (not just antibody polypeptides) against a range of targetantigens is easily applied to any polypeptide repertoire. There arevarious methods for biasing the amino acid distribution at the positionto be varied (including the use of tri-nucleotide mutagenesis, seeWO97/08320), of which the preferred method, due to ease of synthesis, isthe use of conventional degenerate codons. By comparing the amino acidprofile encoded by all combinations of degenerate codons (with single,double, triple and quadruple degeneracy in equal ratios at eachposition) with the natural amino acid use it is possible to calculatethe most representative codon. The codons (AGT)(AGC)T, (AGT)(AGC)C and(AGT)(AGC)(CT)—that is, DVT, DVC and DVY, respectively using IUPACnomenclature—are those closest to the desired amino acid profile: theyencode 22% serine and 11% tyrosine, asparagine, glycine, alanine,aspartate, threonine and cysteine. Preferably, therefore, libraries areconstructed using either the DVT, DVC or DVY codon at each of thediversified positions.

G: Antigens Capable of Increasing Ligand Half-Life

The dual specific ligands according to the invention, in oneconfiguration thereof, are capable of binding to one or more moleculeswhich can increase the half-life of the ligand in vivo. Typically, suchmolecules are polypeptides which occur naturally in vivo and whichresist degradation or removal by endogenous mechanisms which removeunwanted material from the organism. For example, the molecule whichincreases the half-life of the organism may be selected from thefollowing:

Proteins from the extracellular matrix; for example collagen, laminins,integrins and fibronectin. Collagens are the major proteins of theextracellular matrix. About 15 types of collagen molecules are currentlyknown, found in different parts of the body, e.g. type I collagen(accounting for 90% of body collagen) found in bone, skin, tendon,ligaments, cornea, internal organs or type II collagen found incartilage, invertebral disc, notochord, vitreous humour of the eye.

Proteins found in blood, including: Plasma proteins such as fibrin, α-2macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloidprotein A, heptaglobin, profilin, ubiquitin, uteroglobulin andβ-2-microglobulin;

Enzymes and inhibitors such as plasminogen, lysozyme, cystatin C,alpha-1-antitrypsin and pancreatic trypsin inhibitor. Plasminogen is theinactive precursor of the trypsin-like serine protease plasmin. It isnormally found circulating through the blood stream. When plasminogenbecomes activated and is converted to plasmin, it unfolds a potentenzymatic domain that dissolves the fibrinogen fibers that entgangle theblood cells in a blood clot. This is called fibrinolysis.

Immune system proteins, such as IgE, IgG, IgM.

Transport proteins such as retinol binding protein, α-1 microglobulin.

Defensins such as beta-defensin 1, Neutrophil defensins 1, 2 and 3.

Proteins found at the blood brain barrier or in neural tissues, such asmelanocortin receptor, myelin, ascorbate transporter.

Transferrin receptor specific ligand-neuropharmaceutical agent fusionproteins (see U.S. Pat. No. 5,977,307);

brain capillary endothelial cell receptor, transferrin, transferrinreceptor, insulin, insulin-like growth factor 1 (IGF 1) receptor,insulin-like growth factor 2 (IGF 2) receptor, insulin receptor.

Proteins localised to the kidney, such as polycystin, type IV collagen,organic anion transporter K1, Heymann's antigen.

Proteins localised to the liver, for example alcohol dehydrogenase,G250.

Blood coagulation factor X

α1 antitrypsin

HNF 1α

Proteins localised to the lung, such as secretory component (binds IgA).

Proteins localised to the Heart, for example HSP 27. This is associatedwith dilated cardiomyopathy.

Proteins localised to the skin, for example keratin.

Bone specific proteins, such as bone morphogenic proteins (BMPs), whichare a subset of the transforming growth factor β superfamily thatdemonstrate osteogenic activity. Examples include BMP-2, -4, -5, -6, -7(also referred to as osteogenic protein (OP-1) and -8 (OP-2).

Tumour specific proteins, including human trophoblast antigen, herceptinreceptor, oestrogen receptor, cathepsins e.g. cathepsin B (found inliver and spleen).

Disease-specific proteins, such as antigens expressed only on activatedT-cells: including LAG-3 (lymphocyte activation gene), osteoprotegerinligand (OPGL) see Nature 402, 304-309; 1999, OX40 (a member of the TNFreceptor family, expressed on activated T cells and the onlycostimulatory T cell molecule known to be specifically up-regulated inhuman T cell leukaemia virus type-I (HTLV-I)-producing cells.) See JImmunol. 2000 Jul. 1; 165(1): 263-70; Metalloproteases (associated witharthritis/cancers), including CG6512 Drosophila, human paraplegin, humanFtsH, human AFG3L2, murine ftsH; angiogenic growth factors, includingacidic fibroblast growth factor (FGF-1), basic fibroblast growth factor(FGF-2), Vascular endothelial growth factor/vascular permeability factor(VEGF/VPF), transforming growth factor-a (TGF a), tumor necrosisfactor-alpha (TNF-α), angiogenin, interleukin-3 (IL-3), interleukin-8(IL-8), platelet-derived endothelial growth factor (PD-ECGF), placentalgrowth factor (PlGF), midkine platelet-derived growth factor-BB (PDGF),fractalkine.

Stress proteins (heat shock proteins) HSPs are normally foundintracellularly. When they are found extracellularly, it is an indicatorthat a cell has died and spilled out its contents. This unprogrammedcell death (necrosis) only occurs when as a result of trauma, disease orinjury and therefore in vivo, extracellular HSPs trigger a response fromthe immune system that will fight infection and disease. A dual specificligand which binds to extracellular HSP can be localised to a diseasesite.

Proteins involved in Fc transport

Brambell receptor (also known as FcRB)

This Fc receptor has two functions, both of which are potentially usefulfor delivery.

The functions are

(1) The transport of IgG from mother to child across the placenta(2) the protection of IgG from degradation thereby prolonging its serumhalf life of IgG. It is thought that the receptor recycles IgG fromendosome.

See Holliger et al, Nat Biotechnol 1997 July; 15(7):632-6.

Ligands according to the invention may designed to be specific for theabove targets without requiring any increase in or increasing half lifein vivo. For example, ligands according to the invention can be specificfor targets selected from those described above which aretissue-specific, thereby enabling tissue-specific targeting of the dualspecific ligand, or a dAb monomer that binds a tissue-specifictherapeutically relevant target, irrespective of any increase inhalf-life, although this may result. Moreover, where the ligand or dAbmonomer targets kidney or liver, this may redirect the ligand or dAbmonomer to an alternative clearance pathway in vivo (for example, theligand may be directed away from liver clearance to kidney clearance).

As described above, ligands described herein comprising a singlevariable domain as defined herein can be selected to be specific for atarget and preferably may have the added attribute of increasing thehalf life of a target in vivo, though not required. A dual-specificligand may be composed of an antibody heavy chain single variable domainhaving a binding specificity to a first epitope or antigen, and also ofan antibody light chain single variable domain having a bindingspecificity to a second epitope or antigen, where one or both of theantigens can be serum albumin, or one or both of the epitopes is anepitope(s) of serum albumin. In one embodiment, both serum albuminepitopes are the same, in another embodiment, each serum albumin epitopeis different.

In addition to these dual-specific ligands which have the attribute ofincreasing the half life of a target in vivo, other structural forms ofligands are described herein which have or consist of at least onesingle variable domain as defined herein which has the attribute ofincreasing the half life of a target binding ligand in vivo, e.g., bybinding serum albumin. For example, the ligand can consist of, orcontain, a monomer single variable domain as defined herein which bindsserum albumin; or the ligand can be in a form which comprises multiplesingle variable domains as defined herein, where one or more of thesingle variable domains binds serum albumin, i.e., a multimer. Both themultimer and the monomer can further comprise other entities in additionto the one or more single variable domain(s) which binds serum albumin,e.g., in the form of a fusion protein and/or a conjugate. Such a fusionprotein preferably is a single polypeptide chain and can comprise forexample two or more linked single variable domains as defined herein;the linked single variable domains can be identical to each other orthey can be different from each other. Such entities include e.g., oneor more additional single variable domains as defined herein, which havea specificity to an antigen or epitope other than serum albumin, and/orone or more drugs, and/or one or more target binding domains which havea specificity to an antigen or epitope other than serum albumin andwhich are not single variable domains as defined herein. Such a multimermay have multiple valencies with respect to its single variabledomain(s), e.g., univalent, divalent, trivalent, tetravalent. Such amultimer may have the form of an IgG structure or a dual specific ligandas defined herein, as well as other structures such as IgM, IgE, IgD, orIgA, and/or fragments thereof, including but not limited to fragmentssuch as scFv fragments, Fab, Fab′ etc. The ligand can be modified tocontain additional moieties, such as a fusion protein, or a conjugate.

An antibody heavy chain single variable domain of a dual specific ligandor of a monomer ligand or of a multimer ligand as described herein, canspecifically bind serum albumin and comprises an amino acid sequence ofan antibody heavy chain single variable domain. Such an antibody heavychain single variable domain can be selected from, but preferably is notlimited to, one of the following domains: dAb7r20, dAb7r21, dAb7r22,dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30,dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25,Ab7h26, dAb7h27, dAb7h30 and dAb7h31, or a domain with an amino acidsequence that is at least 80% identical thereto, up to and including85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto, and specificallybinds serum albumin. Alternatively, the ligand comprises an antibodysingle variable domain, preferably an antibody heavy chain singlevaraible domain, that competes for binding to serum albumin with one ofthe following domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24,dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,dAb7h30 dAb7h31, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4,dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17,dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9,dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2, or witha domain having an amino acid sequence that is at least 80% identicalthereto, up to and including 85%, 90%, 95%, 96%, 97%, 98%, 99% identicalthereto, and that specifically binds serum albumin. Alternatively, theligand comprises, in addition to the antibody heavy chain singlevariable domain, an antibody light chain single variable domain whichcan specifically bind serum albumin and comprise an amino acid sequenceof an antibody light chain single variable domain. Such an antibodylight chain single variable domain can be selected from, but preferablyis not limited to, one of the following domains: dAb7m12, dAb7m16,dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13,dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2,dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13,dAb7h14, dAb7p1, and dAb7p2, or a domain with an amino acid sequencethat is at least 80% identical thereto, up to and including 85%, 90%,95%, 96%, 97%, 98%, 99% identical thereto, and that specifically bindsserum albumin. Alternatively, the ligand comprises an antibody singlevariable domain, preferably an antibody light chain single variabledomain, that competes for binding to serum albumin with a domain thatcan be selected from, but preferably not limited to, one of thefollowing domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25,dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33,dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30dAb7h31, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18,dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10,dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2, or a domainhaving an amino acid sequence that is at least 80% identical thereto, upto and including 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto,and having binding specificity for serum albumin. In one embodiment, theligand can be an IgG immunoglobulin having any combination of one, ortwo of the above dual specific ligands. In one embodiment, the ligandcan contain one or more monomers of the single variable domains listedabove, where if the ligand contains more than one of these singlevariable domains, the single variable domains can be identical to eachother, or not identical to each other.

In one embodiment, the ligand can be a dual specific ligand which has afirst immunoglobulin single variable domain having a first antigen orepitope binding specificity and a second immunoglobulin single variabledomain having a second antigen or epitope binding specificity, the firstand the second immunoglobulin single variable domains being antibodyheavy chain single variable domains, and where one or both of the firstand second antibody heavy chain single variable domains specificallybinds to serum albumin and has an amino acid sequence of an antibodyheavy chain single variable domain that can be selected from, but ispreferably not limited to, one of the following antibody heavy chainsingle variable domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24,dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,dAb7h30 and dAb7h31, or an amino acid sequence that is at least 80%identical thereto, up to and including 85%, 90%, 95%, 96%, 97%, 98%, or99% identical thereto. One embodiment of such a ligand is a dualspecific ligand which has a first immunoglobulin single variable domainhaving a first antigen or epitope binding specificity and a secondimmunoglobulin single variable domain having a second antigen or epitopebinding specificity, the first and the second immunoglobulin singlevariable domains being antibody heavy chain single variable domains, andwhere one or both of the first and second antibody heavy chain singlevariable domains specifically binds to serum albumin and competes forbinding to serum albumin with a single variable domain which has anamino acid sequence of an antibody single variable domain that can beselected from, but is preferably not limited to, one of the followingantibody single variable domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23,dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31,dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26,dAb7h27, dAb7h30 dAb7h31, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2,or a sequence that is at least 80% identical thereto, or up to andincluding 85%, 90%, 95%, 96%, 97%, 98%, 99% identical thereto. In oneembodiment, the ligand can be an IgG immunoglobulin having anycombination of one or two of the above dual specific ligands. In oneembodiment, the ligand can contain one or more monomers of the singlevariable domains listed above, where if the ligand contains more thanone of these single variable domains, the single variable domains can beidentical to each other, or not identical to each other.

In one embodiment a dual specific ligand has a first immunoglobulinsingle variable domain having a first antigen or epitope bindingspecificity and a second immunoglobulin single variable domain having asecond antigen or epitope binding specificity, the first and the secondimmunoglobulin single variable domains being antibody light chain singlevariable domains, and one or both of the first and second antibody lightchain single variable domains specifically binds to serum albumin andhas an amino acid sequence of an antibody light chain single variabledomain that can be selected from, but is preferably not limited to, oneof the following antibody light chain single variable domains dAb7m12,dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1,dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12,dAb7h13, dAb7h14, dAb7p1, and dAb7p2, or a sequence that is at least 80%identical thereto, or up to and including 85%, 90%, 95%, 96%, 97%, 98%,or 99% identical thereto.

In one embodiment, the ligand can be a dual specific ligand which has afirst immunoglobulin single variable domain having a first antigen orepitope binding specificity and a second immunoglobulin single variabledomain having a second antigen or epitope binding specificity, the firstand the second immunoglobulin single variable domains being antibodylight chain single variable domains, and one or both of the first andsecond antibody light chain single variable domains specifically bindsto serum albumin and competes for binding to serum albumin with anantibody light chain single variable domain which has an amino acidsequence of an antibody single variable domain which can be selectedfrom, but preferably is not limited to, one of the following antibodysingle variable domains: dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24,dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,dAb7h30 dAb7h31, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4,dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17,dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9,dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1 and dAb7p2, or asequence that is at least 80% identical thereto, or up to and including85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

Described herein is a ligand which has one or more antibody heavy chainsingle variable domains where the one or more antibody heavy chainsingle variable domain specifically binds serum albumin and has an aminoacid sequence of an antibody heavy chain single variable domain selectedfrom, but preferably not limited to, that of dAb8, dAb 10, dAb36,dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27,dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22,dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, and asequence that is at least 80% identical thereto, or up to and including85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

Described herein is a ligand which has one or more antibody heavy chainsingle variable domains, where the one or more antibody heavy chainsingle variable domains specifically binds serum albumin and competesfor binding to serum albumin with an antibody single variable domainwhich has an amino acid sequence of an antibody single variable domainselected from, but preferably not limited to, that of one of thefollowing: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23,dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31,dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26,dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15,dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26,dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47,dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1,dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15,dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7,dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, anddAb7p2.

Described herein is a ligand which has an antibody heavy chain singlevariable domain having a binding specificity to a first antigen, orepitope thereof, and an antibody light chain single variable domainhaving a binding specificity to a second antigen, or epitope thereof,where one or both of the first antigen and said second antigen is serumalbumin, and where the antibody heavy chain single variable domainspecifically binds serum albumin and competes for binding to serumalbumin with an antibody single variable domain which has an amino acidsequence of an antibody single variable domain selected from, butpreferably not limited to, the group: dAb8, dAb 10, dAb36, dAb7r20,dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23,Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7,dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22,dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35,dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12,dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1,dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12,dAb7h13, dAb7h14, dAb7p1, dAb7p2, and where the antibody light chainsingle variable domain specifically binds serum albumin and has an aminoacid sequence of an antibody light chain single variable domain selectedfrom, but preferably not limited to, that of the following: dAb2, dAb4,dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21,dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34,dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56,drdAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7,dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2, and a sequence that is atleast 80% identical thereto, or up to and including 85%, 90%, 95%, 96%,97%, 98%, or 99% identical thereto.

Described herein is a ligand which has an antibody heavy chain singlevariable domain having a binding specificity to a first antigen orepitope thereof, and an antibody light chain single variable domainhaving a binding specificity to a second antigen or epitope thereof,wherein one or both of said first antigen and said second antigen isserum albumin, and wherein the antibody heavy chain single variabledomain specifically binds serum albumin albumin and has an amino acidsequence of an antibody heavy chain single variable domain selected frombut preferably not limited to, the group: dAb8, dAb 10, dAb36, dAb7r20,dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28,dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23,Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, and a sequence thatis at least 80% identical thereto, or up to and including 85%, 90%, 95%,96%, 97%, 98%, or 99% identical thereto, and where the antibody lightchain single variable domain specifically binds serum albumin andcompetes for binding to serum albumin with an antibody single variabledomain which comprises an amino acid sequence of an antibody singlevariable domain selected from, but preferably not limited to the group:dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24,dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16,dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27,dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52,dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1 and dAb7p2.

Described herein is a ligand which has one or more antibody heavy chainsingle variable domains having a binding specificity to a first antigenor epitope thereof, and one or more antibody light chain single variabledomains having a binding specificity to a second antigen or epitopethereof, wherein one or both of the first antigen and the second antigenis serum albumin, and wherein the one or more antibody heavy chainsingle variable domains specifically binds serum albumin and competesfor binding to serum albumin with an antibody single variable domainwhich has an amino acid sequence of an antibody single variable domainselected from, but preferably not limited to, the group: dAb8, dAb 10,dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21,dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31,dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18,dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31,dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54,dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18,dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10,dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, dAb7p2, and where the one ormore antibody light chain single variable domains specifically bindsserum albumin and comprises an amino acid sequence of an antibody lightchain single variable domain selected from, but preferably not limitedto, the group: dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16,dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27,dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52,dAb53, dAb54, dAb55, dAb56, drdAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2,and an amino acid sequence that is at least 80% identical thereto, or upto and including 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

Described herein is a ligand which has one or more antibody heavy chainsingle variable domains having a binding specificity to a first antigenor epitope thereof, and one or more antibody light chain single variabledomains having a binding specificity to a second antigen or epitopethereof, where one or both of said first antigen and said second antigenis serum albumin, and where the one or more antibody heavy chain singlevariable domains specifically binds serum albumin albumin and has anamino acid sequence of an antibody heavy chain single variable domainselected from, but preferably not limited to, the group: dAb8, dAb 10,dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21,dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, anda sequence that is at least 80% identical thereto, or up to andincluding 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, andwhere the one or more antibody light chain single variable domainsspecifically binds serum albumin and competes for binding to serumalbumin with an antibody single variable domain which has an amino acidsequence of an antibody single variable domain selected from the group:dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24,dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16,dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27,dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52,dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1 and dAb7p2.

Described herein is a ligand which has one or more antibody light chainsingle variable domains and where the one or more antibody light chainsingle variable domains specifically binds serum albumin and has anamino acid sequence of an antibody light chain single variable domainselected from, but preferably not limited to, the group: dAb2, dAb4,dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21,dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34,dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56,drdAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7,dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,dAb7h12, dAb7h13, dAb7h14, dAb7p1, dAb7p2, and a sequence that is atleast 80% identical thereto, or up to and including 85%, 90%, 95%, 96%,97%, 98%, or 99% identical thereto.

Described herein is a ligand which has one or more antibody light chainsingle variable domains, where the one or more antibody light chainsingle variable domains specifically binds serum albumin and competesfor binding to serum albumin with an antibody single variable domainwhich has an amino acid sequence of an antibody single variable domainselected from, but preferably not limited to, the group: dAb8, dAb 10,dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21,dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31,dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18,dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31,dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54,dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18,dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10,dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2.

Described herein is a ligand which has one or more single variabledomains, where the one or more single variable domains specificallybinds serum albumin and competes for binding to serum albumin with anantibody single variable domain which has an amino acid sequence of anantibody single variable domain selected from, but preferably notlimited to the group: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22,dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30,dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25,Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12,dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24,dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41,dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16,dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13,dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2,dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13,dAb7h14, dAb7p1, and dAb7p2. Preferably, the one or more single variabledomains comprises a scaffold selected from, but preferably not limitedto, the group consisting of CTLA-4, lipocallin, SpA, an Affibody, anavimer, GroEl and fibronectin, and competes for binding to serum albuminwith an antibody single variable domain which has an amino acid sequenceof an antibody single variable domain selected from, but preferably notlimited to the group: dAb8, dAb 10, dAb36, dAb7r20, dAb7r21, dAb7r22,dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30,dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25,Ab7h26, dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12,dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24,dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41,dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16,dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13,dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2,dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13,dAb7h14, dAb7p1, and dAb7p2.

Described herein is a ligand which has a first immunoglobulin singlevariable domain having a first antigen or epitope binding specificityand a second immunoglobulin single variable domain having a secondantigen or epitope binding specificity, where the first and the secondimmunoglobulin single variable domains are antibody heavy chain singlevariable domains, where the first antibody heavy chain single variabledomains specifically binds to serum albumin and has an amino acidsequence of an antibody heavy chain single variable domain selectedfrom, but preferably not limited to, the group: dAb8, dAb 10, dAb36,dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26, dAb7r27,dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21, dAb7h22,dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31, and asequence that is at least 80% identical thereto, or up to and including85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and where thesecond antibody heavy chain single variable domains specifically bindsto serum albumin and competes for binding to serum albumin with anantibody single variable domain which has an amino acid sequence of anantibody single variable domain selected from the group: dAb8, dAb 10,dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21,dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31,dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18,dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31,dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54,dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18,dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10,dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2.

Described herein is a ligand which has a first immunoglobulin singlevariable domain having a first antigen or epitope binding specificityand a second immunoglobulin single variable domain having a secondantigen or epitope binding specificity, where the first and the secondimmunoglobulin single variable domains are antibody light chain singlevariable domains, where the first antibody light chain single variabledomain specifically binds to serum albumin and has an amino acidsequence of an antibody light chain single variable domain selectedfrom, but preferably not limited to, the group: dAb2, dAb4, dAb7, dAb11,dAb12, dAb13, dAb15, dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23,dAb24, dAb25, dAb26, dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38,dAb41, dAb46, dAb47, dAb52, dAb53, dAb54, dAb55, dAb56, drdAb7m12,dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7, dAb7r8,dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19, dAb7h1,dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12,dAb7h13, dAb7h14, dAb7p1, dAb7p2, and a sequence that is at least 80%identical thereto, or up to and including 85%, 90%, 95%, 96%, 97%, 98%,or 99% identical thereto, and where the second antibody light chainsingle variable domain specifically binds to serum albumin and competesfor binding to serum albumin with an antibody single variable domainwhich has an amino acid sequence of an antibody single variable domainselected from, but preferably not limited to, the group: dAb8, dAb 10,dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21,dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30, dAb7h31,dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16, dAb17, dAb18,dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27, dAb30, dAb31,dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52, dAb53, dAb54,dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5,dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18,dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10,dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2.

Embodiments of ligands described supra and herein, also includes thosehaving a structure comprising an IgG structure having any combination ofone, or two of the above dual specific ligands, and/or single variabledomains comprising non-immunoglobulin scaffolds. Such an immunoglobulinstructure can have various combinations of antibody single variabledomains, including an IgG structure that contains four antibody heavychain single variable domains, or an IgG structure that contains fourantibody light chain single variable domains, as well as an IgGstructure that contains two pairs of chains, each pair containing anantibody heavy chain single variable domain and an antibody light chainsingle variable domain. In addition to these IgG structures, the ligandsdescribed herein can contain one or more monomers of a single variabledomain, including but preferably not limited to the single variabledomains listed above, where if the ligand contains more than one ofthese single variable domains, the single variable domains can beidentical to each other, or not identical to each other.

Embodiments of ligands comprising one or more single variable domainsinclude, but preferably are not limited to, the dAbs described herein,dual specific monomers comprising at least one single variable domain,dual specific IgG molecules containing antibody single chain monomers,and multivalent IgG molecules comprising antibody single chain monomersas described herein. These embodiments, can further comprise a bindingsite for a generic ligand. The generic ligand can include, butpreferably is not limited to, protein A, protein L and protein G. Forsuch dual specific ligands, including those in an IgG format, thetarget(s) for each second antigen or epitope binding specificityincludes, but preferably is not limited to, a binding specificity for anantigen which can be characterized in a group selected from cytokines,cytokine receptors, enzymes, enzyme co-factors and DNA binding proteins,and can be selected from, but preferably is not limited to, EPOreceptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGF receptor,ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic, FGF-basic,fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF,G-CSF, GM-CSF, GF-β1, insulin, IFN-γIL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8 (72 a.a.), IL-8 (77 a.a), IL-9, IL-10, IL-11, IL-12, IL-13,IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10keratinocyte growth factor-2 (KGF-2), KGF, Leptin, L1F, Lymphotactin,Mullerian inhibitory substance, monocyte colony inhibitory factor,monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1(MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a), MIG, MLP-1α,MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M,PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β□□ TGF-β, TGF-β2,TNF receptor 1, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1,VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-γ, HCC1, 1-309,HER 1, HER 2, HER3, HER4, CD4, human chemokine receptors CXCR4 or CCR5,non-structural protein type 3 (NS3) from the hepatitis C virus,TNF-alpha, IgE, IFN-gamma, MMP-12, CEA, H. pylori, TB, influenza,Hepatitis E, MMP-12, internalising receptors such as the epidermalgrowth factor receptor (EGFR), ErBb2 receptor on tumor cells, aninternalising cellular receptor, LDL receptor, FGF2 receptor, ErbB2receptor, transferrin receptor, PDGF receptor, VEGF receptor, PsmAr, anextracellular matrix protein, elastin, fibronectin, laminin,α1-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1, Bad,caspase-9, Forkhead, an antigen of Helicobacter pylori, an antigen ofMycobacterium tuberculosis, and an antigen of influenza virus. In such adual-specific ligand, including those dual specific ligands present inan IgG format, one or both single variable domains specifically binds anepitope or antigen with a dissociation constant (Kd) that can beselected from, but is preferably not limited to, 1×10⁻³ M or less,1×10⁻⁴ M or less, 1×10⁻⁵ M or less, 1×10⁻⁶ M or less, 1×10⁻⁷ M or less,1×10⁻⁸ M or less, and 1×10⁻⁹ M or less, as determined, for example, bysurface plasmon resonance. Such a dual-specific ligand, including thosedual specific ligands present in an IgG format, can further contain oneor more entities including, but preferably is not limited to a label, atag and a drug. Such a dual-specific ligand, including those dualspecific ligands present in an IgG format, as well as a multimericligand that contains one or more monomers of the single variable domainslisted above, can be present in a kit, and in a composition, including apharmaceutical composition, containing the dual specific ligand and acarrier thereof.

Similarly, for a ligand comprising one or more single variable domainsas described herein, including a ligand in monomeric form and a ligandin multimeric form as defined supra, the one or more single variabledomains specifically binds an epitope or antigen with a dissociationconstant (Kd) that can be selected from, but is preferably not limitedto, 1×10⁻³ M or less, 1×10⁻⁴ M or less, 1×10⁻⁵ M or less, 1×10⁻⁶ M orless, 1×10⁻⁷ M or less, 1×10⁻⁸ M or less, and 1×10⁻⁹ M or less, asdetermined, for example, by surface plasmon resonance. Such a ligand canfurther contain one or more entities including, but preferably notlimited to a label, a tag and a drug. Such ligand can be present in akit, a composition, including a pharmaceutical composition, containingthe ligand and a carrier thereof.

Percent identity, where recited herein can refer to the percent identityalong the entire stretch of the length of the amino acid or nucleotidesequence. When specified, the percent identity of the amino acid ornucleic acid sequence refers to the percent identity to sequence(s) fromone or more discrete regions of the referenced amino acid or nucleicacid sequence, for instance, along one or more antibody CDR regions,and/or along one or more antibody variable domain framework regions. Forexample, the sequence identity at the amino acid level across one ormore CDRs of a polypeptide can have at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to the aminoacid sequence of corresponding CDRs of an antibody heavy or light chainsingle variable domain. Similarly, the sequence identity at the aminoacid level across one or more framework regions of a polypeptide canhave at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% or higher identity to the amino acid sequence of a correspondingframework of an antibody heavy or light chain single variable domain. Atthe nucleic acid level, the nucleic acid sequence encoding one or moreCDRs of a polypeptide can have at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to thenucleic acid sequence encoding corresponding CDRs of an antibody heavyor light chain single variable domain. At the nucleic acid level, thenucleic acid sequence encoding one or more framework regions of apolypeptide can have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or higher identity to the nucleic acidsequence encoding corresponding framework regions of an antibody heavyor light chain single variable domain, respectively. The frameworkregions (FW) are preferably from an antibody framework region, such asthe human V3-23[DP47]JH4B heavy or the human kappa light chain DPK9/JK1.If the framework region(s) is that found in the human V3-23[DP47]/JH4Bheavy chain V region, the percent identity can be targeted to itsframework regions and/or preferably to one or more of the CDR regions asillustrated in FIG. 24. If the framework is that found in the humanDPK9/JK1 light chain V region, the percent identity can be compared toits referenced framework regions and/or preferably to one or more of theCDR regions as illustrated in FIG. 24.

The CDRs are preferably those of an antibody variable domain,preferably, but not limited to those of antibody single variabledomains.

In some embodiments, the structural characteristic of percent identityis coupled to a functional aspect. For instance, in some embodiments, anucleic acid sequence or amino acid sequence with less than 100%identity to a referenced nucleic acid or amino acid sequence is alsorequired to display at least one functional aspect of the referenceamino acid sequence or of the amino acid sequence encoded by thereferenced nucleic acid. In other embodiments, a nucleic acid sequenceor amino acid sequence with less than 100% identity to a referencednucleic acid or amino acid sequence, respectively, is also required todisplay at least one functional aspect of the reference amino acidsequence or of the amino acid sequence encoded by the referenced nucleicacid, but that functional characteristic can be slightly altered, e.g.,confer an increased affinity to a specified antigen relative to that ofthe reference.

H: Use of Multispecific Ligands According to the Second Configuration ofthe Invention

Multispecific ligands according to the method of the secondconfiguration of the present invention may be employed in in vivotherapeutic and prophylactic applications, in vitro and in vivodiagnostic applications, in vitro assay and reagent applications, andthe like. For example antibody molecules may be used in antibody basedassay techniques, such as ELISA techniques, according to methods knownto those skilled in the art.

As alluded to above, the multispecific ligands according to theinvention are of use in diagnostic, prophylactic and therapeuticprocedures. Multispecific antibodies according to the invention are ofuse diagnostically in Western analysis and in situ protein detection bystandard immunohistochemical procedures; for use in these applications,the ligands may be labelled in accordance with techniques known to theart. In addition, such antibody polypeptides may be used preparativelyin affinity chromatography procedures, when complexed to achromatographic support, such as a resin. All such techniques are wellknown to one of skill in the art.

Diagnostic uses of the closed conformation multispecific ligandsaccording to the invention include homogenous assays for analytes whichexploit the ability of closed conformation multispecific ligands to bindtwo targets in competition, such that two targets cannot bindsimultaneously (a closed conformation), or alternatively their abilityto bind two targets simultaneously (an open conformation).

A true homogenous immunoassay format has been avidly sought bymanufacturers of diagnostics and research assay systems used in drugdiscovery and development. The main diagnostics markets include humantesting in hospitals, doctor's offices and clinics, commercial referencelaboratories, blood banks, and the home, non-human diagnostics (forexample food testing, water testing, environmental testing, bio-defence,and veterinary testing), and finally research (including drugdevelopment; basic research and academic research).

At present all these markets utilise immunoassay systems that are builtaround chemiluminescent, ELISA, fluorescence or in rare casesradio-immunoassay technologies. Each of these assay formats requires aseparation step (separating bound from un-bound reagents). In somecases, several separation steps are required. Adding these additionalsteps adds reagents and automation, takes time, and affects the ultimateoutcome of the assays. In human diagnostics, the separation step may beautomated, which masks the problem, but does not remove it. Therobotics, additional reagents, additional incubation times, and the likeadd considerable cost and complexity. In drug development, such as highthroughput screening, where literally millions of samples are tested atonce, with very low levels of test molecule, adding additionalseparation steps can eliminate the ability to perform a screen. However,avoiding the separation creates too much noise in the read out. Thus,there is a need for a true homogenous format that provides sensitivitiesat the range obtainable from present assay formats.

Advantageously, an assay possesses fully quantitative read-outs withhigh sensitivity and a large dynamic range. Sensitivity is an importantrequirement, as is reducing the amount of sample required. Both of thesefeatures are features that a homogenous system offers. This is veryimportant in point of care testing, and in drug development wheresamples are precious. Heterogenous systems, as currently available inthe art, require large quantities of sample and expensive reagents

Applications for homogenous assays include cancer testing, where thebiggest assay is that for Prostate Specific Antigen, used in screeningmen for prostate cancer. Other applications include fertility testing,which provides a series of tests for women attempting to conceiveincluding beta-hcg for pregnancy. Tests for infectious diseases,including hepatitis, HIV, rubella, and other viruses and microorganismsand sexually transmitted diseases. Tests are used by blood banks,especially tests for HIV, hepatitis A, B, C, non A non B. Therapeuticdrug monitoring tests include monitoring levels of prescribed drugs inpatients for efficacy and to avoid toxicity, for example digoxin forarrhythmia, and phenobarbital levels in psychotic cases; theophyllinefor asthma.

Diagnostic tests are moreover useful in abused drug testing, such astesting for cocaine, marijuana and the like. Metabolic tests are usedfor measuring thyroid function, anaemia and other physiologicaldisorders and functions.

The homogenous immunoassay format is moreover useful in the manufactureof standard clinical chemistry assays. The inclusion of immunoassays andchemistry assays on the same instrument is highly advantageous indiagnostic testing. Suitable chemical assays include tests for glucose,cholesterol, potassium, and the like.

A further major application for homogenous immunoassays is drugdiscovery and development: high throughput screening includes testingcombinatorial chemistry libraries versus targets in ultra high volume.Signal is detected, and positive groups then split into smaller groups,and eventually tested in cells and then animals. Homogenous assays maybe used in all these types of test. In drug development, especiallyanimal studies and clinical trials heavy use of immunoassays is made.Homogenous assays greatly accelerate and simplify these procedures.Other Applications include food and beverage testing: testing meat andother foods for E. coli, salmonella, etc; water testing, includingtesting at water plants for all types of contaminants including E. coli;and veterinary testing.

In a broad embodiment, the invention provides a binding assay comprisinga detectable agent which is bound to a closed conformation multispecificligand according to the invention, and whose detectable properties arealtered by the binding of an analyte to said closed conformationmultispecific ligand. Such an assay may be configured in severaldifferent ways, each exploiting the above properties of closedconformation multispecific ligands.

The assay relies on the direct or indirect displacement of an agent bythe analyte, resulting in a change in the detectable properties of theagent. For example, where the agent is an enzyme which is capable ofcatalysing a reaction which has a detectable end-point, said enzyme canbe bound by the ligand such as to obstruct its active site, therebyinactivating the enzyme. The analyte, which is also bound by the closedconformation multispecific ligand, displaces the enzyme, rendering itactive through freeing of the active site. The enzyme is then able toreact with a substrate, to give rise to a detectable event. In analternative embodiment, the ligand may bind the enzyme outside of theactive site, influencing the conformation of the enzyme and thusaltering its activity. For example, the structure of the active site maybe constrained by the binding of the ligand, or the binding of cofactorsnecessary for activity may be prevented.

The physical implementation of the assay may take any form known in theart. For example, the closed conformation multispecific ligand/enzymecomplex may be provided on a test strip; the substrate may be providedin a different region of the test strip, and a solvent containing theanalyte allowed to migrate through the ligand/enzyme complex, displacingthe enzyme, and carrying it to the substrate region to produce a signal.Alternatively, the ligand/enzyme complex may be provided on a test stickor other solid phase, and dipped into an analyte/substrate solution,releasing enzyme into the solution in response to the presence ofanalyte.

Since each molecule of analyte potentially releases one enzyme molecule,the assay is quantitative, with the strength of the signal generated ina given time being dependent on the concentration of analyte in thesolution.

Further configurations using the analyte in a closed conformation arepossible. For example, the closed conformation multispecific ligand maybe configured to bind an enzyme in an allosteric site, therebyactivating the enzyme. In such an embodiment, the enzyme is active inthe absence of analyte. Addition of the analyte displaces the enzyme andremoves allosteric activation, thus inactivating the enzyme.

In the context of the above embodiments which employ enzyme activity asa measure of the analyte concentration, activation or inactivation ofthe enzyme refers to an increase or decrease in the activity of theenzyme, measured as the ability of the enzyme to catalyse asignal-generating reaction. For example, the enzyme may catalyse theconversion of an undetectable substrate to a detectable form thereof.For example, horseradish peroxidase is widely used in the art togetherwith chromogenic or chemiluminescent substrates, which are availablecommercially. The level of increase or decrease of the activity of theenzyme may between 10% and 100%, such as 20%, 30%, 40%, 50%, 60%, 70%,80% or 90%; in the case of an increase in activity, the increase may bemore than 100%, i.e. 200%, 300%, 500% or more, or may not be measurableas a percentage if the baseline activity of the inhibited enzyme isundetectable.

In a further configuration, the closed conformation multispecific ligandmay bind the substrate of an enzyme/substrate pair, rather than theenzyme. The substrate is therefore unavailable to the enzyme untilreleased from the closed conformation multispecific ligand throughbinding of the analyte. The implementations for this configuration areas for the configurations which bind enzyme.

Moreover, the assay may be configured to bind a fluorescent molecule,such as a fluorescein or another fluorophore, in a conformation suchthat the fluorescence is quenched on binding to the ligand. In thiscase, binding of the analyte to the ligand will displace the fluorescentmolecule, thus producing a signal. Alternatives to fluorescent moleculeswhich are useful in the present invention include luminescent agents,such as luciferin/luciferase, and chromogenic agents, including agentscommonly used in immunoassays such as HRP.

Therapeutic and prophylactic uses of multispecific ligands preparedaccording to the invention involve the administration of ligandsaccording to the invention to a recipient mammal, such as a human.Multi-specificity can allow antibodies to bind to multimeric antigenwith great avidity. Multispecific ligands can allow the cross-linking oftwo antigens, for example in recruiting cytotoxic T-cells to mediate thekilling of tumour cell lines.

Substantially pure ligands or binding proteins thereof, for example dAbmonomers, of at least 90 to 95% homogeneity are preferred foradministration to a mammal, and 98 to 99% or more homogeneity is mostpreferred for pharmaceutical uses, especially when the mammal is ahuman. Once purified, partially or to homogeneity as desired, theligands may be used diagnostically or therapeutically (includingextracorporeally) or in developing and performing assay procedures,immunofluorescent stainings and the like (Lefkovite and Pernis, (1979and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

The ligands or binding proteins thereof, for example dAb monomers, ofthe present invention will typically find use in preventing, suppressingor treating inflammatory states, allergic hypersensitivity, cancer,bacterial or viral infection, and autoimmune disorders (which include,but are preferably not limited to, Type I diabetes, asthma, multiplesclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn'sdisease and myasthenia gravis).

In the instant application, the term “prevention” involvesadministration of the protective composition prior to the induction ofthe disease. “Suppression” refers to administration of the compositionafter an inductive event, but prior to the clinical appearance of thedisease. “Treatment” involves administration of the protectivecomposition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe antibodies or binding proteins thereof in protecting against ortreating the disease are available. Methods for the testing of systemiclupus erythematosus (SLE) in susceptible mice are known in the art(Knight et al. (1978) J. Exp. Med., 147: 1653; Reinersten et al. (1978)New Eng. J. Med., 299: 515). Myasthenia Gravis (MG) is tested in SJL/Jfemale mice by inducing the disease with soluble AchR protein fromanother species (Lindstrom et al. (1988) Adv. Immunol., 42: 233).Arthritis is induced in a susceptible strain of mice by injection ofType II collagen (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233). Amodel by which adjuvant arthritis is induced in susceptible rats byinjection of mycobacterial heat shock protein has been described (VanEden et al. (1988) Nature, 331: 171). Thyroiditis is induced in mice byadministration of thyroglobulin as described (Maron et al. (1980) J.Exp. Med., 152: 1115). Insulin dependent diabetes mellitus (IDDM) occursnaturally or can be induced in certain strains of mice such as thosedescribed by Kanasawa et al. (1984) Diabetologia, 27: 113. EAE in mouseand rat serves as a model for MS in human. In this model, thedemyelinating disease is induced by administration of myelin basicprotein (see Paterson (1986) Textbook of Immunopathology, Mischer etal., eds., Grune and Stratton, New York, pp. 179-213; McFarlin et al.(1973) Science, 179: 478: and Satoh et al. (1987) J. Immunol., 138:179).

A ligand comprising a single variable domain, or composition thereof,which specifically binds vWF, e.g., human vWF, a vWF A1 domain, the A1domain of activated vWF, or the vWF A3 domain, may further comprise athrombolytic agent. This thrombolytic agent may be non-covalently orcovalently attached to a single variable domain, in particular to anantibody single variable domain, via covalent or non-covalent means asknown to one of skill in the art. Non-covalent means include via aprotein interaction such as biotin/strepavidin, or via animmunoconjugate. Alternatively, the thrombolytic agent may beadministered simultaneously, separately or sequentially with respect toa ligand that consists of or comprises a single variable domain thatbinds vWF or a vWF domain as described above, or a composition thereof.Thrombolytic agents according to the invention may include, for example,staphylokinase, tissue plasminogen activator, streptokinase, singlechain streptokinase, urokinase and acyl plasminogen streptokinasecomplex.

Also described herein are invasive medical devices coated with a singlevariable domain, or a ligand comprising a single variable domain, or acomposition thereof, or a single varable domain resulting from ascreening method described herein. Non-limiting examples of devicesinclude surgical tubing, occlusion devices, prosthetic devices.Application for said devices include surgical procedures which require amodulation of platelet-mediated aggregation around the site of invasion(e.g. a device coated with a single variable domain which specificallybinds vWF) or a modulation of inflammation (e.g. a device coated with asingle variable domain which specifically binds TNF alpha).

Generally, the present ligands will be utilised in purified formtogether with pharmacologically appropriate carriers. Typically, thesecarriers include aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, any including saline and/or buffered media. Parenteralvehicles include sodium chloride solution, Ringer's dextrose, dextroseand sodium chloride and lactated Ringer's. Suitablephysiologically-acceptable adjuvants, if necessary to keep a polypeptidecomplex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude various immunotherapeutic drugs, such as cylcosporine,methotrexate, adriamycin or cisplatinum, and immunotoxins.Pharmaceutical compositions can include “cocktails” of various cytotoxicor other agents in conjunction with the ligands of the presentinvention, or even combinations of ligands according to the presentinvention having different specificities, such as ligands selected usingdifferent target antigens or epitopes, whether or not they are pooledprior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected ligands thereof of the invention can beadministered to any patient in accordance with standard techniques. Theadministration can be by any appropriate mode, including parenterally,intravenously, intramuscularly, intraperitoneally, transdermally, viathe pulmonary route, or also, appropriately, by direct infusion with acatheter. The dosage and frequency of administration will depend on theage, sex and condition of the patient, concurrent administration ofother drugs, counterindications and other parameters to be taken intoaccount by the clinician.

The ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins andart-known lyophilisation and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilisationand reconstitution can lead to varying degrees of antibody activity loss(e.g. with conventional immunoglobulins, IgM antibodies tend to havegreater activity loss than IgG antibodies) and that use levels may haveto be adjusted upward to compensate.

The compositions containing the present ligands or a cocktail thereofcan be administered for prophylactic and/or therapeutic treatments. Incertain therapeutic applications, an adequate amount to accomplish atleast partial inhibition, suppression, modulation, killing, or someother measurable parameter, of a population of selected cells is definedas a “therapeutically-effective dose”. Amounts needed to achieve thisdosage will depend upon the severity of the disease and the generalstate of the patient's own immune system, but generally range from 0.005to 5.0 mg of ligand, e.g. antibody, receptor (e.g. a T-cell receptor) orbinding protein thereof per kilogram of body weight, with doses of 0.05to 2.0 mg/kg/dose being more commonly used. For prophylacticapplications, compositions containing the present ligands or cocktailsthereof may also be administered in similar or slightly lower dosages.

Treatment performed using the compositions described herein isconsidered “effective” if one or more symptoms is reduced (e.g., by atleast 10% or at least one point on a clinical assessment scale),relative to such symptoms present before treatment, or relative to suchsymptoms in an individual (human or model animal) not treated with suchcomposition. Symptoms will obviously vary depending upon the disease ordisorder targeted, but can be measured by an ordinarily skilledclinician or technician. Such symptoms can be measured, for example, bymonitoring the level of one or more biochemical indicators of thedisease or disorder (e.g., levels of an enzyme or metabolite correlatedwith the disease, affected cell numbers, etc.), by monitoring physicalmanifestations (e.g., inflammation, tumor size, etc.), or by an acceptedclinical assessment scale, for example, the Expanded Disability StatusScale (for multiple sclerosis), the Irvine Inflammatory Bowel DiseaseQuestionnaire (32 point assessment evaluates quality of life withrespect to bowel function, systemic symptoms, social function andemotional status—score ranges from 32 to 224, with higher scoresindicating a better quality of life), the Quality of Life RheumatoidArthritis Scale, or other accepted clinical assessment scale as known inthe field. A sustained (e.g., one day or more, preferably longer)reduction in disease or disorder symptoms by at least 10% or by one ormore points on a given clinical scale is indicative of “effective”treatment. Similarly, prophylaxis performed using a composition asdescribed herein is “effective” if the onset or severity of one or moresymptoms is delayed, reduced or abolished relative to such symptoms in asimilar individual (human or animal model) not treated with thecomposition.

A composition containing a ligand or cocktail thereof according to thepresent invention may be utilised in prophylactic and therapeuticsettings to aid in the alteration, inactivation, killing or removal of aselect target cell population in a mammal. In addition, the selectedrepertoires of polypeptides described herein may be usedextracorporeally or in vitro selectively to kill, deplete or otherwiseeffectively remove a target cell population from a heterogeneouscollection of cells. Blood from a mammal may be combinedextracorporeally with the ligands, e.g. antibodies, cell-surfacereceptors or binding proteins thereof whereby the undesired cells arekilled or otherwise removed from the blood for return to the mammal inaccordance with standard techniques.

I: Use of Half-Life Enhanced Dual-Specific Ligands According to theInvention

Dual-specific ligands according to the method of the present invention,as well a ligands comprising one or more single variable domains asdefined herein, may be employed in in vivo therapeutic and prophylacticapplications, in vivo diagnostic applications and the like.

Therapeutic and prophylactic uses of dual-specific ligands preparedaccording to the invention, as well a ligands comprising one or moresingle variable domains as defined herein, involve the administration ofligands according to the invention to a recipient mammal, such as ahuman. Dual specific antibodies according to the invention as well aligands comprising one or more single variable domains as definedherein, comprise at least one specificity for a half-life enhancingmolecule; one or more further specificities may be directed againsttarget molecules. For example, a dual-specific IgG may be specific forfour epitopes, one of which is on a half-life enhancing molecule.Dual-specificity as well as tri-specificity as well as high valencies,can allow ligands comprising at least one single variable domain, tobind to multimeric antigen with great avidity. Dual-specific antibodiescan allow the cross-linking of two antigens, for example in recruitingcytotoxic T-cells to mediate the killing of tumour cell lines.

Substantially pure dual-specific ligands according to the method of thepresent invention, as well a ligands comprising one or more singlevariable domains as defined herein, or binding proteins thereof, such assingle variable domain monomers (i.e. dAb monomers), of at least 90 to95% homogeneity are preferred for administration to a mammal, and 98 to99% or more homogeneity is most preferred for pharmaceutical uses,especially when the mammal is a human. Once purified, partially or tohomogeneity as desired, the ligands may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes Iand II, Academic Press, NY).

Dual-specific ligands according to the method of the present invention,as well a ligands comprising one or more single variable domains asdefined herein, will typically find use in preventing, suppressing ortreating inflammatory states, allergic hypersensitivity, cancer,bacterial or viral infection, and autoimmune disorders (which include,but are preferably not limited to, Type I diabetes, multiple sclerosis,rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease andmyasthenia gravis).

In the instant application, the term “prevention” involvesadministration of the protective composition prior to the induction ofthe disease. “Suppression” refers to administration of the compositionafter an inductive event, but prior to the clinical appearance of thedisease. “Treatment” involves administration of the protectivecomposition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe dual specific ligands in protecting against or treating the diseaseare available. Methods for the testing of systemic lupus erythematosus(SLE) in susceptible mice are known in the art (Knight et al. (1978) J.Exp. Med., 147: 1653; Reinersten et al. (1978) New Eng. J. Med., 299:515). Myasthenia Gravis (MG) is tested in SJL/J female mice by inducingthe disease with soluble AchR protein from another species (Lindstrom etal. (1988) Adv. Immunol., 42: 233). Arthritis is induced in asusceptible strain of mice by injection of Type II collagen (Stuart etal. (1984) Ann. Rev. Immunol., 42: 233). A model by which adjuvantarthritis is induced in susceptible rats by injection of mycobacterialheat shock protein has been described (Van Eden et al. (1988) Nature,331: 171). Thyroiditis is induced in mice by administration ofthyroglobulin as described (Maron et al. (1980) J. Exp. Med., 152:1115). Insulin dependent diabetes mellitus (IDDM) occurs naturally orcan be induced in certain strains of mice such as those described byKanasawa et al. (1984) Diabetologia, 27: 113. EAE in mouse and ratserves as a model for MS in human. In this model, the demyelinatingdisease is induced by administration of myelin basic protein (seePaterson (1986) Textbook of Immunopathology, Mischer et al., eds., Gruneand Stratton, New York, pp. 179-213; McFarlin et al. (1973) Science,179: 478: and Satoh et al. (1987)J. Immunol., 138: 179).

Dual specific ligands according to the invention and dAb monomers ableto bind to extracellular targets involved in endocytosis (e.g. Clathrin)enable dual specific ligands to be endocytosed, enabling anotherspecificity able to bind to an intracellular target to be delivered toan intracellular environment. This strategy requires a dual specificligand with physical properties that enable it to remain functionalinside the cell. Alternatively, if the final destination intracellularcompartment is oxidising, a well folding ligand may not need to bedisulphide free.

Generally, the present dual specific ligands will be utilised inpurified form together with pharmacologically appropriate carriers.Typically, these carriers include aqueous or alcoholic/aqueoussolutions, emulsions or suspensions, any including saline and/orbuffered media. Parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.Suitable physiologically-acceptable adjuvants, if necessary to keep apolypeptide complex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude various immunotherapeutic drugs, such as cylcosporine,methotrexate, adriamycin or cisplatinum, and immunotoxins.Pharmaceutical compositions can include “cocktails” of various cytotoxicor other agents in conjunction with the ligands of the presentinvention.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the ligands of the invention can be administered to anypatient in accordance with standard techniques. The administration canbe by any appropriate mode, including parenterally, intravenously,intramuscularly, intraperitoneally, transdermally, via the pulmonaryroute, or also, appropriately, by direct infusion with a catheter. Thedosage and frequency of administration will depend on the age, sex andcondition of the patient, concurrent administration of other drugs,counterindications and other parameters to be taken into account by theclinician.

The ligands of the invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins andart-known lyophilisation and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilisationand reconstitution can lead to varying degrees of antibody activity loss(e.g. with conventional immunoglobulins, IgM antibodies tend to havegreater activity loss than IgG antibodies) and that use levels may haveto be adjusted upward to compensate.

The compositions containing the present ligands or a cocktail thereofcan be administered for prophylactic and/or therapeutic treatments. Incertain therapeutic applications, an adequate amount to accomplish atleast partial inhibition, suppression, modulation, killing, or someother measurable parameter, of a population of selected cells is definedas a “therapeutically-effective dose”. Amounts needed to achieve thisdosage will depend upon the severity of the disease and the generalstate of the patient's own immune system, but generally range from 0.005to 5.0 mg of ligand per kilogram of body weight, with doses of 0.05 to2.0 mg/kg/dose being more commonly used. For prophylactic applications,compositions containing the present ligands or cocktails thereof mayalso be administered in similar or slightly lower dosages.

A composition containing a ligand according to the present invention maybe utilised in prophylactic and therapeutic settings to aid in thealteration, inactivation, killing or removal of a select target cellpopulation in a mammal.

In addition, the selected repertoires of polypeptides described hereinmay be used extracorporeally or in vitro selectively to kill, deplete orotherwise effectively remove a target cell population from aheterogeneous collection of cells. Blood from a mammal may be combinedextracorporeally with the ligands, e.g., antibodies, cell surfacereceptors or binding proteins thereof, whereby the undesired cells arekilled or otherwise removed from the blood for return to the mammal inaccordance with standard techniques.

Selection and characterization of ligands comprising a single variabledomain for binding to serum albumin from a range of species

A ligand can comprise one or more single variable domains, e.g.,immunoglobulin single variable domains and/or non-immunoglobulin singlevariable domains, where at least one of the single variable domainsspecifically binds to serum albumin from human, as well as fromnon-human species. In one embodiment, the single variable domainspecifically binds only serum albumin which is endogenous to a human. Inanother embodiment, the single variable domain specifically binds serumalbumin from a non-human species. Alternatively, the single variabledomain specifically binds both serum albumin which is endogenous to ahuman, as well as serum albumin which is endogenous to one or more nonhuman species. As a nonlimiting example, such a single variable domaincan specifically bind serum albumin endogenous to both human andcynomolgus, or serum albumin endogenous to both human and rat, or serumalbumin from both human and mouse, or serum albumin from both human andpig. Alternatively, the single variable domain specifically binds to twoor more serum albumin from two or more non-human species. As usedherein, serum albumin can be expressed by a gene endogenous to aspecies, i.e. natural serum albumin, and/or by a recombinant equivalentthereof. In one embodiment, the serum albumin includes fragments,analogs and derivatives of natural and recombinant serum albumin. Suchfragments of serum albumin include fragments containing domain I, domainII, and/or domain III, or combinations of one or two or more of each ofdomains I, II and III of serum albumin, preferably human serum albumin.Domain II of serum albumin is preferred as a target for the singlevariable domain as defined herein. Other preferred combinations areDomain I and Domain II; Domain I and Domain III; Domain II and DomainIII; and Domain I alone; Domain II alone; and Domain III alone; andDomain I and Domain II and Domain III. In one embodiment, the serumalbumin is recombinant serum albumin exogenously expressed in anon-human host, such as an animal host, or a unicellular host such asyeast or bacteria.

The species from which the serum albumin is endogenous includes anyspecies which expresses endogenous serum albumin, including, butpreferably not limited to, the species of human, mouse, murine, rat,cynomolgus, porcine, dog, cat, horse, goat, and hamster. In someinstances serum albumin endogenous to camel or lama are excluded.

The single variable domain can be an immunoglobulin single variabledomain, including but preferably not limited to an antibody heavy chainsingle variable domain, an antibody VHH heavy chain single variabledomain, a human antibody heavy chain single variable domain, a human VH3heavy chain single variable domain, an antibody light chain singlevariable domain, a human antibody light chain single variable domain, ahuman antibody kappa light chain single variable domain, and/or a humanlambda light chain single variable domain.

The single variable domain which specifically binds to serum albumin canbe a single variable domain comprising an immunoglobulin scaffold or anon-immunoglobulin scaffold. The serum albumin binding, single variabledomain can comprise one or two or three of CDR1, CDR2 and CDR3 from anantibody variable domain, preferably from a single variable domain,where the CDR region(s) is provided on a non-immunoglobulin scaffold,such as CTLA-4, lipocallin, staphylococcal protein A (SPA), GroEL andfibronectin, transferrin (commercially available from Biorexis), anAvimer™ and an Affibody™ scaffold. Alternatively, the serum albuminbinding, non-immunoglobulin single variable domain can contain neitheran antibody CDR region(s) nor a complete binding domain from anantibody. Alternatively, the serum albumin binding, single variabledomain(s), can be single variable domains which comprise one or two orthree of any of CDR1, CDR2 and CDR3 from an antibody variable domain,preferably a single variable domain; these CDR regions can be providedon a heavy or a light chain antibody framework region. Frameworksinclude, for example, VH frameworks, such as VH3 (including DP47, DP38and DP45) and VHH frameworks described supra, as well as VL frameworks,including Vkappa (such as DPK9), and Vlambda frameworks. In someembodiments, the variable domain comprises at least one human frameworkregion having an amino acid sequence encoded by a human germ lineantibody gene segment, or an amino acid sequence comprising up to 5amino acid differences relative to the amino acid sequence encoded by ahuman germ line antibody gene segment. In other embodiments, thevariable domain comprises four human framework regions, FW1, FW2, FW2and FW4, having amino acid sequences encoded by a human germ lineantibody gene segment, or the amino acid sequences of FW1, FW2, FW3 andFW4 collectively containing up to 10 amino acid differences relative tothe amino acid sequences encoded by the human germ line antibody genesegment. In one embodiment, all three CDR regions are provided on eitheran immunoglobulin scaffold (e.g., heavy chain or light chain antibodyscaffold) or a non-immunoglobulin scaffold as defined herein, either ofwhich can be non-human, synthetic, semi-synthetic. Alternatively, anycombination of one, two or all three of CDR1, CDR2 and/or CDR3 regionsare provided on either the immunoglobulin scaffold or thenon-immunoglobulin scaffold, for example, either the CDR3 region alone,or the CDR2 and CDR3 regions together, or the CDR1 and CDR2 are providedon either the immunoglobulin scaffold or the non-immunoglobulinscaffold. Suitable scaffolds and techniques for such CDR grafting willbe clear to the skilled person and are well known in the art, see forexample U.S. application Ser. No. 07/180,370, WO 01/27160, EP 0 605 522,EP 0 460 167, U.S. application Ser. No. 07/054,297, Nicaise et al.,Protein Science (2004), 13:1882-1891; Ewert et al., Methods, 2004October; 34(2):184-199; Kettleborough et al., Protein Eng. 1991 October;4(7): 773-783; O'Brien and Jones, Methods Mol. Biol. 2003: 207: 81-100;and Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et al., J.Mol. Biol. 2005 Sep. 23; 352(3):597-607, and the further referencescited therein.

The ligands can comprise one or more of such single variable domainswhich specifically bind serum albumin, preferably comprising at leastone single variable domain which specifically binds both serum albuminwhich is endogenous to humans and at least one additional serum albuminwhich is endogenous to a non-human species. In one embodiment, thissingle variable domain specifically binds to serum albumin which isendogenous to human with a Kd value which is within 10 fold of the Kdvalue with which it specifically binds (i.e. cross reacts with) to atleast one serum albumin which is endogenous to a non-human species.Alternatively this single variable domain specifically binds to serumalbumin which is endogenous to human with a Kd value which is within 15,20, 25, 30, 50 or up to approximately 100 fold of the Kd value withwhich it specifically binds (i.e. cross reacts with) to at least oneserum albumin which is endogenous to a non-human species. In someembodiments the Kd can range from 300 nM to about 5 pM. In otherembodiments, the single variable domain specifically binds to serumalbumin with a K_(off) of at least 5×10⁻¹, S⁻¹, 5×10⁻² S⁻¹, 5×10⁻³ S⁻¹,5×10⁻⁴ S⁻¹, 5×10⁻⁵ S⁻¹, 5×10⁻⁶ S⁻¹, 5×10⁻⁷ S⁻¹, 5×10⁻⁸ S⁻¹, 5×10⁻⁹ S⁻¹,5×10⁻¹⁹ S⁻¹, or less, preferably with a K_(off) ranging from 1×10′ S⁻¹to 1×10⁻⁸ S⁻¹.

In one embodiment, this single variable domain specifically binds toserum albumin which is endogenous to a first non-human species with a Kdvalue which is within 10 fold of the Kd value with which it specificallybinds to (i.e. cross reacts to) at least one serum albumin which isendogenous to a second non-human species. Alternatively, this singlevariable domain specifically binds to serum albumin which is endogenousto the first non-human species with a Kd value which is within 15, 20,25, 30, 50 or up to approximately 100 fold of the Kd value with which itspecifically binds to (i.e. cross reacts to) to at least one serumalbumin which is endogenous to the second non-human species. In someembodiments, the Kd can range from 300 nM to about 5 pM. In otherembodiments, the single variable domain specifically binds to serumalbumin with a K_(off) of at least 5×10⁻¹, S⁻¹ 5×10⁻² S⁻¹, 5×10⁻³ S⁻¹,5×10⁻⁴ S⁻¹, 5×10⁻⁵ S⁻¹, 5×10⁻⁶ S⁻¹, 5×10⁻⁷ S⁻¹, 5×10⁻⁸ S⁻¹, 5×10⁻⁹ S⁻¹,5×10⁻¹⁰ S⁻¹, or less, preferably with a K_(off) ranging from 1×10⁻⁶ S⁻¹to 1×10⁻⁸ S⁻¹.

For example, such a ligand can include an immunoglobulin single variabledomain, where the immunoglobulin single variable domain specificallybinds to human serum albumin and mouse serum albumin, and where the Tbeta half life of the ligand is substantially the same as the T betahalf life of mouse serum albumin in a mouse host. In one version of sucha ligand, the epitope binding domain contains a non-immunoglobulinscaffold which specifically binds to human serum albumin and mouse serumalbumin, and wherein the T beta half life of the ligand is substantiallythe same as the T beta half life of mouse serum albumin in a mouse host.The phrase “substantially the same” means that the ligand has a T betahalf life in a mouse host that is at least 50% that of mouse serumalbumin in a mouse host, that is at least 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 105%,110%, 125%, and up to 150% that of the T beta half life of mouse serumalbumin in a mouse host. The non-immunoglobulin scaffold can optionallyinclude fragments of an antibody single variable domain, such as one ormore of the CDR regions of an antibody variable domain, including anantibody single variable domain that has a T beta half life in a humanhost that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%,94%, 95%, 96%, 97%, 98%, 99%, 101%, 102%, 105%, 110%, 125%, or up to150% that of the T beta half life of human serum albumin in a humanhost.

For example, one embodiment is a single variable domain, where thesingle variable domain specifically binds to human serum albumin and ratserum albumin, and where the T beta half life of the ligand issubstantially the same as the T beta half life of rat serum albumin in arat host. In one version of such a ligand, the single variable bindingdomain contains a non-immunoglobulin scaffold which specifically bindsto human serum albumin and rat serum albumin, and wherein the T betahalf life of the ligand is substantially the same as the T beta halflife of rat serum albumin in a rat host. The phrase “substantially thesame” means that the ligand has a T beta half life in a rat host that isat least 50% that of rat serum albumin in a rat host, that is up to 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%,100%, 101%, 102%, 105%, 110%, 125%, up to 150% that of the T beta halflife of rat serum albumin in a rat host. The non-immunoglobulin scaffoldcan optionally include fragments of an antibody single variable domain,such as one or more of the CDR regions of an antibody variable domain.

For example, a ligand can include an immunoglobulin single variabledomain, where the immunoglobulin single variable domain specificallybinds to human serum albumin and porcine serum albumin, and where the Tbeta half life of the ligand is substantially the same as the T betahalf life of porcine serum albumin in a porcine host. In one version ofa ligand, the epitope binding domain contains a non-immunoglobulinscaffold which specifically binds to human serum albumin and porcineserum albumin, and wherein the T beta half life of the ligand issubstantially the same as the T beta half life of porcine serum albuminin a porcine host. The phrase “substantially the same” means that theligand has a T beta half life in a porcine host that is at least 50%that of porcine serum albumin in a porcine host, that is up to 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,101%, 102%, 105%, 110%, 125%, up to 150% that of the T beta half life ofporcine serum albumin in a porcine host. The non-immunoglobulin scaffoldcan optionally include fragments of an antibody single variable domain,such as one or more of the CDR regions of an antibody variable domain,including an antibody single variable domain.

For example, a ligand can include an immunoglobulin single variabledomain, where the immunoglobulin single variable domain specificallybinds to human serum albumin and cynomolgus serum albumin, and where theT beta half life of the ligand is substantially the same as the T betahalf life of cynomolgus serum albumin in a cynomolgus host. In oneversion of a ligand, the domain that binds serum albumin contains anon-immunoglobulin scaffold which specifically binds to human serumalbumin and cynomolgus serum albumin, and wherein the T beta half lifeof the ligand is substantially the same as the T beta half life ofcynomolgus serum albumin in a cynomolgus host. The phrase “substantiallythe same” means that the ligand has a T beta half life in a cynomolgushost that is at least 50% that of cynomolgus serum albumin in acynomolgus host, that is up to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 105%, 110%, 125%,or up to 150% that of the T beta half life of cynomolgus serum albuminin a cynomolgus host.

The non-immunoglobulin scaffold can optionally include fragments of anantibody single variable domain, such as one or more of the CDR regionsof an antibody variable domain.

In one embodiment, a ligand and/or dual specific ligand contains asingle variable domain which specifically binds to serum albumin that isendogenous to human, has a T beta half life in a human host that is atleast 50% that of human serum albumin in a human host, up to 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,101%, 102%, 105%, 110%, 125% or up to 150% that of the T beta half lifeof human serum albumin in a human host. In a preferred embodiment, thesingle variable domain which specifically binds to serum albumin that isendogenous to a non-human, has a T beta half life in its respectivenon-human host that is at least 50% that of the non human serum albuminin its respective non-human host, up to 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 105%,110%, 125%, or up to 150% that of the T beta half life of the non-humanserum albumin in its respective non-human host. In a preferredembodiment, the single variable domain which specifically binds to serumalbumin that is endogenous to human, and which also specifically bindsspecifically to serum albumin from at least one non-human species, has aT beta half life in a human host that is up to 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 101%, 102%, 105%,110%, 125%, or up to 150% of human serum albumin in a human host, and aT beta half life in the non-human host that is up to 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%,105%, 110%, 125%, or up to 150% of the non-human serum albumin in itsrespective non-human host. In some embodiments, the T beta half life ofthe single variable domain which specifically binds to serum albumin canrange from as low as 2 hours up to and including 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days,4 days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18days, up to as high as 21 days or more. In a human host, as well as anon-human host such as a porcine, cynomulgus, rat, murine, mouse host,the T beta half life of the single variable domain which specificallybinds to serum albumin can range from as low as 2 hours up to andincluding 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours,10 hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22hours, 1 day, 2 days, 3 days, 4 days, 4 days, 6 days, 8 days, 10 days,12 days, 14 days, 16 days, 18 days, up to as high as 21 days, or more.Other preferred T beta half lives of a ligand comprising a singlevariable domain which specifically binds to serum albumin include: in amonkey host from about 3 to about 5, 6, 7, or 8 days, including from aslow as 2 hours, up to and including 3 hours, 4 hours, 5 hours, 6 hours,7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14 hours, 16hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 4days, 6 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, up toas high as 21 days. In a rat or mouse host, the T beta half life of thesingle variable domain which specifically binds to serum albumin canrange from as low as 40 hours to as high as about 75 hours, and includesas low as 2 hours up to and including 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18days, up to as high as 21 days.

The single variable domain which specifically binds to serum albuminincludes Vkappa single variable domains, selected from, but preferablynot limited to DOM7h-9 DOM7h-1, DOM7h-8, DOM7h-9, DOM7h-11, DOM7h-12,DOM7h-13 and DOM7h-14. DOM7r-3 and DOM7r-16, and/or those domains whichcompete for binding serum albumin, preferably human serum albumin, withthe single variable domains selected from, but preferably not limitedto, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24, dAb7r25, dAb7r26,dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32, dAb7r33, dAb7h21,dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30 dAb7h31,dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3, dAb7r4, dAb7r5, dAb7r7,dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16, dAb7r17, dAb7r18, dAb7r19,dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8, dAb7h9, dAb7h10, dAb7h11,dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2. The single variabledomain which specifically binds to serum albumin can be an antibodyheavy chain single variable domain, in particular, human VH3, or VHH. Anafore-mentioned single variable domain may also additionallyspecifically bind human serum albumin with a K_(off) of at least 5×10¹,S⁻¹, 5×10⁻² S⁻¹, 5×10⁻³ S⁻¹, 5×10⁻⁴ S⁻¹, 5×10⁻⁵ S⁻¹, 5×10⁻⁶ S⁻¹ 5×10⁻⁷S⁻¹, 5×10⁻⁸ S⁻¹, 5×10⁻⁹ S⁻¹ 5×10⁻¹⁰ S⁻¹, or less, preferably with aK_(off) ranging from 1×10⁻⁶ S⁻¹ to 1×10⁻⁸ S⁻¹. Single variable domainsthat specifically bind human serum albumin and a serum albumin that isendogenous to a non human species, can further bind a serum albumin thatis endogenous to a third, fourth, fifth, sixth, seventh, eighth, ninthor tenth non human species. In one nonlimiting embodiment, the singlevariable domain which specifically binds to human serum albumin and ratserum albumin, further specifically binds to cynomolgus serum albumin.In another nonlimiting embodiment, the single variable domain whichspecifically binds to human serum albumin and mouse serum albumin,further specifically binds to cynomolgus serum albumin.

As described herein, a ligand which contains one single variable domain(monomer) or more than one single variable domains (multimer, fusionprotein, conjugate, and dual specific ligand as defined herein) whichspecifically binds to serum albumin, can further comprise one or moreentities selected from, but preferably not limited to a label, a tag, anadditional single variable domain, a dAb, an antibody, and antibodyfragment, a marker and a drug. One or more of these entities can belocated at either the COOH terminus or at the N terminus or at both theN terminus and the COOH terminus of the ligand comprising the singlevariable domain, (either immunoglobulin or non-immunoglobulin singlevariable domain). One or more of these entities can be located at eitherthe COOH terminus, or the N terminus, or both the N terminus and theCOOH terminus of the single variable domain which specifically bindsserum albumin of the ligand which contains one single variable domain(monomer) or more than one single variable domains (multimer, fusionprotein, conjugate, and dual specific ligand as defined herein).Non-limiting examples of tags which can be positioned at one or both ofthese termini include a HA, his or a myc tag. The entities, includingone or more tags, labels and drugs, can be bound to the ligand whichcontains one single variable domain (monomer) or more than one singlevariable domain (multimer, fusion protein, conjugate, and dual specificligand as defined herein), which binds serum albumin, either directly orthrough linkers as described in a separate section below.

A ligand which contains one single variable domain (monomer) or morethan one single variable domains (multimer, fusion protein, conjugate,and dual specific ligand as defined herein) which specifically binds toserum albumin, or which specifically binds both human serum albumin andat least one non-human serum albumin, can specifically bind to one ormore of Domain I, and/or Domain II and/or domain III of human serumalbumin, as described further below. In addition to containing one ormore single variable domains, (for example, a serum albumin bindingimmunoglobulin single variable domain or a serum albumin bindingnon-immunoglobulin single variable domain) which specifically binds to aserum albumin, such as human serum albumin, or which specifically bindsboth human serum albumin and at least one non-human serum albumin, theligand can contain one or more additional domains capable ofspecifically binding an antigen and/or epitope other than serum albumin,the antigen or epitope being selected from the group consisting of anyanimal protein, including cytokines, and/or antigens derived frommicroorganisms, pathogens, unicellular organisms, insects, viruses,algae and plants. These one or more additional domain(s) which bind amoiety other than serum albumin can be a non-immunoglobulin bindingdomain, a non-immunoglobulin single variable domain, and/or animmunoglobulin single variable domain.

In some embodiments, a dual specific ligand which contains one or moresingle variable domains (either an immunoglobulin single variable domainor a non-immunoglobulin single variable domain) which specifically bindsto a serum albumin, such as human serum albumin, or which specificallybinds both human serum albumin and at least one non-human serum albumin,can be composed of (a) the single variable domain that specificallybinds serum albumin and a single variable domain that specifically bindsa ligand other than serum albumin, both of the single variable domainsbeing a heavy chain single variable domain; or (b) the single variabledomain that specifically binds serum albumin and a single variabledomain that specifically binds a ligand other than serum albumin, bothof the single variable domains being a light chain single variabledomain; or (c) the single variable domain that specifically binds serumalbumin is a heavy chain single variable domain, and the single variabledomain that specifically binds an antigen other than serum albumin is alight chain single variable domain; or (d) the single variable domainthat specifically binds serum albumin is a light chain single variabledomains, and the single variable domain that specifically binds anantigen other than serum albumin is a heavy chain single variabledomain.

Also encompassed herein is an isolated nucleic acid encoding any of theligands described herein, e.g., a ligand which contains one singlevariable domain (monomer) or more than one single variable domains(e.g., multimer, fusion protein, conjugate, and dual specific ligand asdefined herein) which specifically binds to serum albumin, or whichspecifically binds both human serum albumin and at least one non-humanserum albumin, or functionally active fragments thereof. Alsoencompassed herein is a vector and/or an expression vector thereof, ahost cell comprising the vector, e.g., a plant or animal cell and/orcell line transformed with a vector, a method of expressing and/orproducing one or more ligands which contains one single variable domain(monomer) or more than one single variable domains (e.g., multimer,fusion protein, conjugate, and dual specific ligand as defined herein)which specifically binds to serum albumin, or fragment(s) thereofencoded by said vectors, including in some instances culturing the hostcell so that the one or more ligands or fragments thereof are expressedand optionally recovering the ligand which contains one single variabledomain (monomer) or more than one single variable domains (e.g.,multimer, fusion protein, conjugate, and dual specific ligand as definedherein) which specifically binds to serum albumin, from the host cellculture medium. Also encompassed are methods of contacting a liganddescribed herein with serum albumin, including serum albumin and/ornon-human serum albumin(s), and/or one or more targets other than serumalbumin, where the targets include biologically active molecules, andinclude animal proteins, cytokines as listed above, and include methodswhere the contacting is in vitro as well as administering any of theligands described herein to an individual host animal or cell in vivoand/or ex vivo. Preferably, administering ligands described herein whichcomprises a single variable domain (immunoglobulin ornon-immunoglobulin) directed to serum albumin and/or non-human serumalbumin(s), and one or more domains directed to one or more targetsother than serum albumin, will increase the half life, including the Tbeta half life, of the anti-target ligand. Nucleic acid moleculesencoding the single domain containing ligands or fragments thereof,including functional fragments thereof, are described herein. Vectorsencoding the nucleic acid molecules, including but preferably notlimited to expression vectors, are described herein, as are host cellsfrom a cell line or organism containing one or more of these expressionvectors. Also described are methods of producing any the single domaincontaining ligands, including, but preferably not limited to any of theaforementioned nucleic acids, vectors and host cells.

Epitope Mapping of Serum Albumin

Serum albumins from mammalian species have a similar structure,containing three predominate domains with a similar folding anddisulphide bonding pattern, as highlighted in FIG. 25. The protein isbelieved to have arisen from two tandem duplication events, andsubsequent diversification of residues.

The structure of human serum albumin has been solved by X-raycrystallography, with/without a variety of bound ligands:

-   -   Atomic structure and chemistry of human serum albumin. He X M,        Carter D C.    -   Nature. 1992; 358: 209-15. Erratum in: Nature 1993; 364: 362.    -   Atomic structure and chemistry of human serum albumin. He X M,        Carter D C; J Mol Biol. 2001; 314: 955-60.    -   Crystal structures of human serum albumin complexed with        monounsaturated and polyunsaturated fatty acids. Petitpas I,        Grune T, Bhattacharya A A, Curry S.; J Biol Chem. 2001; 276:        22804-9.

Human serum albumin has been shown to be a heart shaped molecule. Theindividual domains, termed I, II and III, are predominantly helical, andare each composed of two sub-domains, termed IA, IB, IIA, 2B, IIIA, andIIIB. They are linked by flexible, random coils.

Described herein is a ligand which contains one or more single variabledomains which specifically binds to Domain II of human serum albumin.The single variable domain can be a VH antibody single variable domain.The single variable domain can be a VHH antibody single variable domain.The VH single variable domain can be a VH3 single variable domain. TheVH3 single variable domain can be a human VH3 single variable domain.The ligand can alternatively, or additionally include a single variabledomain which is a VKappa antibody single variable domain, including oneof the following: DOM7h-1, DOM7h-8, DOM7h-9, DOM7h-11, DOM7h-12,DOM7h-13, DOM7h-14. DOM7r-3 and DOM7r-16, or a VKappa antibody singlevariable domain having domain having an amino acid sequence of about80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higheridentity thereto.

The antibody single variable domain can include a set of four Kabatframework regions (FRs), which are encoded by antibody VH, preferably aVH3, framework germ line antibody gene segments. The VH3 framework isselected from the group consisting of DP47, DP38 and DP45. The antibodysingle variable domain can include a set of four Kabat framework regions(FRs) which are encoded by an antibody V_(L) framework, preferably aVKappa framework, germline antibody gene segment. Preferably, the Kappaframework is DPK9.

The ligand which contains one or more single variable domains whichspecifically bind to Domain II of human serum albumin can furtherinclude one or more domains capable of specifically binding a moietyother than serum albumin, and can further comprise one or more entitiesincluding one or more of a label, a tag and a drug. The one or moredomains capable of specifically binding a moiety other than serumalbumin can be an immunoglobulin single variable domain. Also describedherein is a ligand which contains one or more single variable domainswhich specifically binds to Domain II of human serum albumin, the domainincluding a non-immunoglobulin scaffold and CDR1, CDR2 and/or CDR3regions, or where at least one of the CDR1, CDR2 and/or CDR3 regions isfrom a single variable domain of an antibody single variable domain thatbinds Domain II of human serum albumin. Non-immunoglobulin scaffoldsinclude, but preferably are not limited to, CTLA-4, lipocallin,staphylococcal protein A (SPA), Affibody™, Avimers™, GroEL andfibronectin.

The ligand which contains one or more single variable domains whichspecifically binds to Domain II of human serum albumin includes thosedomains which specifically bind human serum albumin with a Kd of lessthan or equal to 300 nM. The ligand which contains one or more singlevariable domains which specifically binds to Domain II of human serumalbumin can further comprise one or more entities including one or moreof a label, a tag and a drug. The tag can include one or more ofC-terminal HA or myc tags or N terminal HA or myc tags.

The ligand which contains one or more single variable domains whichspecifically binds to Domain II of human serum albumin, and which canfurther include one or more domains capable of specifically binding amoiety other than serum albumin, and which can optionally furthercomprise one or more entities including one or more of a label, a tagand a drug, can bind, through at least one of its single variabledomains, an antigen including, but preferably not limited to a cytokinereceptor, EPO receptor, ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGFreceptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, EpoR, FGF-acidic,FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C),GDNF, G-CSF, GM-CSF, GF-β1, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a), IL-9,IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibinα, Inhibin β, IP-10 keratinocyte growth factor-2 (KGF-2), KGF, Leptin,L1F, Lymphotactin, Mullerian inhibitory substance, monocyte colonyinhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.),MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69a.a), MIG, MLP-1α, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitorfactor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3,NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α,SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β2, TGF-β3,tumor necrosis factor (TNF), TNF-α, TNF receptor 1, TNF receptor II,TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3,GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER 1, HER 2, HER3 and HER4,CD4, human chemokine receptors CXCR4 or CCR5, non-structural proteintype 3 (NS3) from the hepatitis C virus, TNF-alpha, IgE, IFN-gamma,MMP-12, CEA, H. pylori, TB, influenza, Hepatitis E, MMP-12,internalising receptors that are over-expressed on certain cells, suchas the epidermal growth factor receptor (EGFR), ErBb2 receptor on tumorcells, an internalising cellular receptor, LDL receptor, FGF2 receptor,ErbB2 receptor, transferrin receptor, PDGF receptor, VEGF receptor,PsmAr, an extracellular matrix protein, elastin, fibronectin, laminin,al-antitrypsin, tissue factor protease inhibitor, PDK1, GSK1, Bad,caspase-9, Forkhead, an of an antigen of Helicobacter pylori, an antigenof Mycobacterium tuberculosis, and an antigen of influenza virus.

The ligand which contains one or more single variable domains whichspecifically binds to Domain II of human serum albumin, and which canfurther include one or more domains capable of specifically binding amoiety other than serum albumin, is minimally a dual specific ligand,which can have one of the following structures: (a) each said singlevariable domain that specifically binds to Domain II of serum albuminand said single variable domain that specifically binds a moiety otherthan serum albumin, is an antibody heavy chain single variable domain;or (b) each said single variable domain that specifically binds toDomain II of serum albumin and said single variable domain thatspecifically binds a moiety other than serum albumin, is an antibodylight chain single variable domain; or (c) said single variable domainthat specifically binds to Domain II of serum albumin is an antibodyheavy chain single variable domain, and said single variable domain thatspecifically binds an antigen other than serum albumin is an antibodylight chain single variable domain; or (d) said single variable domainthat specifically binds to Domain II of serum albumin is an antibodylight chain single variable domain, and said single variable domain thatspecifically binds an antigen other than serum albumin is an antibodyheavy chain single variable domain. Nucleic acid molecules encoding anyligands or fragments thereof, including functional fragments thereof,described herein, vectors including but preferably not limited toexpression vectors, and host cells of any type cell line or organism,containing one or more of these expression vectors is included, and/orare methods of producing any ligands, including, but preferably notlimited to any the aforementioned nucleic acids, vectors and host cells.

Serum albumin has a long serum half-life compared with other serumproteins, together with a positive relationship between serumconcentration and fractional catabolic rates (i.e. the higher theconcentration of SA, the higher the amount degraded), a property that itshares with IgG. It has recently emerged that both IgG and serum albuminshare a recycling mechanism, mediated by the neonatal Fc receptor FcRn.FcRn is a type I MHC family member, composed of a heterodimer of themembrane anchored FCRGT chain, and non-membrane-bound beta-2microglobulin. Mouse knockout mutants of either FcRn or beta-2microglobulin express no functional FcRn, and exhibit an increasedbiosynthesis rate of serum albumin (˜20% increase), and an increasedcatabolism of serum albumin, leading to a 40% lower serum level of serumalbumin, with a shorter half-life (Chaudhry et al 2005). In humans,mutations in beta-2 microglobulin have been shown give much reducedfunctional FcRn levels and ultimately to IgG deficiency andhypoalbuminaemia, characterised by a reduced serum half-life of HSA(Wani et al 2006, PNAS).

Though not wishing to be bound by theory, the proposed mechanism forFcRn-mediated salvage is as follows:

1. Plasma proteins are pinocytosed by cells of the endothelium liningall blood vessels, and perhaps pinocytotically active cells of theextravascular compartment. This is a non-specific step, and all proteinsin circulation will be taken up. FcRn has a very low affinity foralbumin (and IgGs) at serum pH, around pH 7.4.

2. Once pinocytosed, the vesicle formed acidifies to pH 5.0. Under acidconditions, FcRn has a higher affinity for albumin, and binds albumin,and also IgG. Albumin and IgG are thus bound to the FcRn receptor. FcRnbinds human serum albumin at a site on Domain III, via a distinct sitefrom that which binds IgG.

3. A sorting event occurs, by which the majority of non-receptor boundproteins are sorted into an endosome, where most proteins will betargeted for degradation. The receptor bound albumin and IgG are sortedinto a vesicle targeted for the cell surface, and thus spared fromdegradation.

4. The cell surface targeted vesicle then either fuses with the cellsurface, or briefly fuses with the cell membrane. Under theseconditions, the pH of the endosome increases to approach pH 7.4, theFcRn affinity for albumin is reduced, and albumin is released back intothe circulation.

We can therefore define a clear set of desirable parameters for any SAbinding protein to have maximum half life. These parameters can beclearly exemplified using the serum albumin salvage receptor FcRn as amodel, although will also apply to other receptors mediating a prolongedhalf life.

-   -   The affinity of the serum albumin binding will preferably be        such that the SA binding protein does not dissociate from        albumin while undergoing glomerular filtration in the kidney,        thus minimising loss to the urine, and/or    -   The binding to SA will preferably not have a detrimental effect        on the binding of serum albumin to any receptors responsible for        the maintenance of serum albumin levels in the circulation, as        this would inhibit recycling, and hence reduce the half-life of        both the serum albumin and the SA binder. Thus SA binding dAbs        preferably bind a distinct epitope from that bound by FcRn on        HSA domain III, and the SA/dAb complex preferably is also        capable of engaging FcRn, and/or    -   The binding to SA will preferably be maintained under the        conditions under which the receptor and bound SA/SA binder        complex are sorted or recycled. Endosomal pH has been shown to        approach pH 5.0, therefore stable binding of the dAb to serum        albumin at both pH7.4 and pH 5.0 is desirable.

As illustrated in Example 15 below, the majority of dAbs bind to the 2nddomain of HSA and are therefore not expected to compete with binding ofhuman serum albumin to FcRn. Two dAbs (DOM7h-27 and DOM7h-30) bind toDomain III.

An anti-SA DAb that retains sufficient affinity for SA in a pH range of7.4 to 5.0.

In addition to affinity for SA, as well as in the absence of competitionwith the formation of SA:FcRn complexes, the serum-albumin-specific dAbswill preferably maintain affinity to SA within a pH range from pH 7.4 inthe serum to pH 5.0 in the endosome to obtain full benefit of theFcRn-mediated salvage pathway.

In this pH range, only histidine residues and amino acid side-chainswith perturbed pKa are likely to change their protonation state. Ifamino acid side-chains make a significant contribution to the bindingenergy of the complex, one could expect that a pH shift from one extremeto the other extreme in the range could result in lowering the bindingaffinity of the complex. Though not wishing to be bound by theory, thisin turn would result in increasing the likelihood that the SA-specificdAb enters in the degradation pathway rather than being rescued throughthe FcRn-mediated salvage pathway.

Thus, for a SA binding AlbudAb™ (a dAb which specifically binds serumalbumin), it is desirable to select one where the bindingcharacteristics to serum albumin do not significantly change with pH (inthe range of 5.0 to 7.4). A straightforward method to ensure this wouldbe to analyze the amino acid sequences of the anti-SA dAbs for theabsence of histidine residues in the CDRs. As shown below, severalselection procedures for such a property can be envisaged:

For example, a first selection round is performed with the ‘naïve’ dAbphage repertoire using immobilized human serum albumin in conditionswhere the pH of the buffer is at pH 7.4 (e.g. PBS). The recovered andamplified phage population is then submitted to a second round ofselection where the incubation buffer is at pH 5.0. The alternation ofbuffers and pHs are optionally repeated in further rounds in order tomaintain selection pressure for dAb binding to HSA at both pHs.

In a second example, all selection rounds are performed with the ‘naïve’dAb phage repertoire using immobilised human serum albumin in conditionswhere the pH of the buffer is at pH 7.4 (e.g. PBS). However, just afterwashing away unbound phage with PBS (or PBS supplemented with Tween) andprior to elution of bound phage, there is added an additionalwash/incubation step at pH 5.0 for a prolonged period of time (e.g. upto 4 hours). During this period, phage displaying dAbs that are unableto bind SA at pH 5.0 (but able to bind at pH 7.4) are detached from theimmobilised SA. After a second series of wash steps (at pH 5.0 with(out)Tween, bound phage is recovered and analysed.

In a third example, all selection rounds are performed with the ‘naïve’dAb phage repertoire using immobilized human serum albumin in conditionswhere the pH of the buffer is at pH 7.4 (e.g. PBS). Best dAb candidates(i.e. able to bind at pH 7.4 and pH 5.0) are then identified byscreening. Typically, the genes encoding dAbs are recovered from thepooled selected phage, subcloned into an expression vector that directsthe soluble dAb in the supernatant of E. coli cultures. Individualclones are picked, grown separately in the wells of microtiter plates,and induced for expression. Supernatants (or purified dAbs) are thendirectly loaded onto a Biacore chip to identify those dAbs displayingaffinity for the immobilised serum albumin. Each supernatant is screenedfor binding (mainly the off-rate trace of the sensorgram) to HSA inconditions where the ‘running’ buffer is either at pH 7.4 or at pH 5.0.It should be noted that screening of dAb binding on the Biacore wouldalso be used as a preferred method to identify best leads from the twoabove examples.

Described herein is a ligand comprising a single variable domain asdefined herein, where the single variable domain specifically bindsserum albumin both at a natural serum pH, and at an intracellularvesicle pH. The natural serum pH is about 7 (e.g., 7.4), and whereinsaid intracellular vesicle pH can range from about 4.8 to 5.2, or can beat a pH of about 5. In one embodiment, the single variable domain canspecifically bind serum albumin with a pH range of about 7 to 5, or canbe at a pH of 7.4. Though not wishing to be bound by theory, a furthercharacteristic of this ligand is that the its single variable domainthat specifically binds serum albumin does not substantially dissociatefrom serum albumin while undergoing glomerular filtration in the kidney.Though not wishing to be bound by theory, a further characteristic ofthis ligand is that its single variable domain that specifically bindsserum albumin does not substantially interfere with the binding of FcRnto the serum albumin. This single variable domain can be an antibodysingle variable domain; the antibody single variable domain can be aV_(H)3 domain and/or the antibody single variable domain can be a Vkappa domain. This single variable domain can comprise anon-immunoglobulin scaffold, e.g., CTLA-4, lipocallin, SpA, Affibody™,GroEL, Avimer™ or fibronectin scaffolds, and can contain one or more ofCDR1, CDR2 and/or CDR3 from an antibody single variable domain thatpreferably, though not necessarily, specifically binds serum albumin.The single variable domain(s) of this ligand, can specifically bindhuman serum albumin, and/or including serum albumin from one or morespecies, e.g., human, rat, monkey, procine, rabbit, hamster, mouse orgoat. The intracellular compartment can be any intracellular compartmentof any cell of any animal, including an endosomal compartment orintracellular vesicle or a budding vesicle. The endosomal compartmentcan have a pH of about 5, or 5.0. The ligands described herein cancontain one or more single variable domains including immunoglobulinand/or non-immunoglobulin domains where the binding of serum albumin tothe single variable domain does not substantially competitively inhibitthe binding of FcRn to serum albumin. These one or more singularvariable domains can preferably specifically bind serum albumin with anequilibrium dissociation constant (Kd) of less than or equal to 300 nM.

Described herein is a method for selecting for a ligand comprising asingle variable domain, which contains one single variable domain(monomer), or more than one single variable domains (e.g., multimer,fusion protein, conjugate, and dual specific ligand as defined herein)which specifically binds to serum albumin, where the single variabledomain specifically binds human serum albumin at a natural serum pH, andwhere the single variable domain does not competitively inhibit thebinding of human serum albumin to FcRn, and where the single variabledomain specifically binds human serum albumin at a pH of anintracellular compartment, comprising the steps of: (A) selecting forligands comprising a single variable domain which does not bind theregions of human serum albumin that bind FcRn, (B) from the ligandsselected in step (A), selecting for ligands comprising a single variabledomain which binds serum albumin at said natural serum pH. (C) selectingthe ligands selected in step (B) for those which bind serum albumin atthe pH of said intracellular compartment. Alternatively steps (A) and(B) can be reversed as follows: (A) selecting ligands comprising asingle variable domain which binds human serum albumin at said naturalserum pH, (B) from the ligands selected in (A), selecting ligandscomprising a single variable domain which binds human serum albuminoutside the regions of HSA that bind FcRn, and (C) from the ligandsselected in step (B), selecting for those which bind serum albumin atsaid pH of said intracellular compartment. Also described is a methodfor selecting for a ligand comprising a single variable domain, wherethe single variable domain specifically binds human serum albumin at anatural serum pH, wherein the single variable domain does notcompetitively inhibit the binding of human serum albumin to FcRn, andwhere the single variable domain specifically binds human serum albuminat a pH of an intracellular compartment, comprising the steps of: (A)selecting for ligands comprising a single variable domain which does notbind the regions of human serum albumin that bind FcRn, (B) from step(A) selecting for ligands comprising a single variable domain whichbinds serum albumin at said natural serum pH, and (C) geneticallymodifying the single variable domain of step (B) such that it bindsserum albumin at said pH of said intracellular compartment.Alternatively steps (A) and (B) can be reversed as follows: (A),selecting for ligands comprising a single variable domain which bindsserum albumin at said natural serum pH, (B) from the ligands selected in(A), selecting ligands comprising a single variable domain which doesnot bind the regions of human serum albumin that bind FcRn, and (C)genetically modifying the single variable domain of step (B) such thatit binds serum albumin at said pH of said intracellular compartment.

An assay to determine if a single variable domain does not competitivelyinhibit the binding of human serum albumin to FcRn: A competitionBiacore experiment can be used to determine if a single variable domaincompetitively inhibits the binding of serum albumin to a FcRn. Oneexperimental protocol for such an example is as follows. After coating aCM5 sensor chip (Biacore AB) at 25° C. with approximately 1100 resonanceunits (RUs) of a purified FcRn at pH 7.4, human serum albumin (HSA), isinjected over the antigen surface at a single concentration (e.g., 1 um)alone, and in combination with a dilution series of mixtures, eachmixture having HSA and increasing amounts of the single variable domainin question. The resulting binding RUs are determined for the HSA aloneand each of the HSA/single variable domain mixtures. By comparing thebound RUs of HSA alone with the bound RUs of HSA+single variable domain,one will be able to see whether the FcRn competes with the singlevariable domain to bind HSA. If it does compete, then as the singlevariable domain concentration in solution is increased, the RUs of HSAbound to FcRn will decrease. If there is no competition, then adding thesingle variable domain will have no impact on how much HSA binds toFcRn. This competition assay can optionally be repeated at pH 5.0 for asingle variable domain which binds HAS at pH 5.0 in order to determineif the single variable domain competitively inhibits the binding ofserum albumin to a FcRn at pH 5.0.

These ligands which have a single variable domain, which contains onesingle variable domain (monomer) or more than one single variabledomains (e.g., multimer, fusion protein, conjugate, and dual specificligand as defined herein) which specifically binds to serum albumin,where the single variable domain specifically binds serum albumin bothat a natural serum pH, and at an intracellular vesicle pH, can furthercomprise at least one additional single variable domain, where eachadditional single variable domain specifically binds an antigen otherthan serum albumin at a natural serum pH, but does not bind the antigenat an intracellular vesicle pH. The natural serum pH is about 7.4, andthe pH of said intracellular vesicle ranges from about 4.8 to 5.2, andin some embodiments, the pH of said intracellular vesicle is about 5.

A method based on the above ideas, includes the use of a bispecificbinder with affinity for a serum albumin to prolong half-life and anaffinity to a desired target antigen, as described above, to direct abound antigen for degradation, or recycling. As described above, a serumalbumin binding moiety is selected, such that binding is of highaffinity at pH 5.0, such that the molecule would be sorted fornon-degradation in the endosome by an FcRn mediated process. A desiredtarget antigen binding moiety is then selected using a similar techniqueas described above, except that, instead of selecting for high affinitybinding at pH 7.4 and pH 5 as described above, selection for highaffinity binding at pH 7.4 is performed, and low or zero affinity forthe target antigen at pH 5. One way to achieve this is by selecting formoieties with histidines in the contact surface. A fusion proteinbetween the 2 molecules is then made by conventional molecular biologytechniques, either by chemical derivitization and crosslinking, or bygenetic fusion. The result is an increase in potency of a given AlbudAb™(a dAb which specifically binds serum albumin) in vivo, by designing aSA binding dAb that binds SA at pH 5, while having a partner dAb thatbinds a ligand, which has low or zero affinity at pH 5. Though notwishing to be bound by theory, upon endosomal recycling, the targetmolecule will be released, and targeted to a degradative endosome anddegraded, while the AlbudAb™ (a dAb which specifically binds serumalbumin) is recycled to bind a fresh ligand via FcRn mediated recycling.This method offers a key advantage over PEGylated molecules or otherhalf life extension technologies, where this pathway is not availablefor regeneration. Presumably in these cases, the bound ligand just sitson the PEGylated moiety and occupies it.

Described herein is a method of directing an antigen for degradationcomprising administering a ligand which has at least one single variabledomain, where the single variable domain specifically binds serumalbumin both at a natural serum pH, and at an intracellular vesicle pH,and which further has at least one additional single variable domain,wherein the single variable domain specifically binds an antigen otherthan serum albumin at a natural serum pH, but does not bind said antigenat an intracellular vesicle pH, thus targeting the antigen other thanserum albumin for degradation. Also described herein is, a ligandfurther comprising at least one additional single variable domain,wherein said single variable domain specifically binds an antigen otherthan serum albumin at a natural serum pH, but does not bind said antigenat an intracellular vesicle pH.

Selecting dAbs In Vitro in the Presence of Metabolites

Encompassed by the ligands described herein, is a ligand comprising asingle variable domain, which contains one single variable domain(monomer) or more than one single variable domains (e.g., multimer,fusion protein, conjugate, and dual specific ligand as defined herein)which specifically binds to serum albumin, where the single variabledomain specifically binds human serum albumin, and where specificbinding of serum albumin by the single variable domain is notessentially blocked by binding of drugs and/or metabolites and/or smallmolecules to one or more sites on serum albumin. The one or more siteson human serum albumin include Sudlow site 1 and Sudlow site 2. The oneor more sites can be located on any combination of one or more domainsof human serum albumin selected from the group consisting of domain I,domain II and domain III.

Encompassed by the ligands described herein, is a ligand comprising asingle variable domain, which contains one single variable domain(monomer) or more than one single variable domains (e.g., multimer,fusion protein, conjugate, and dual specific ligand as defined herein)which specifically binds to serum albumin, where the single variabledomain specifically binds human serum albumin, and where specificbinding of serum albumin by said single variable domain does not alterthe binding characteristics of serum albumin for drugs and/ormetabolites and/or small molecule bound to SA. In one embodiment thesingle variable domain of the ligand binds serum albumin in both thepresence and/or absence of a drug, metabolite or other small molecule.And in another embodiment, the specific binding of serum albumin by saidsingle variable domain does not substantially alter the bindingcharacteristics of serum albumin for drugs and/or metabolites and/orsmall molecules bound to SA naturally in vivo, including, but preferablynot limited to those drugs and/or metabolites and/or small moleculesdescribed in Fasano et al. (2005) 57(12):787-96. The extraordinaryligand binding properties of human serum albumin, and Bertucci, C. etal. (2002) 9(15):1463-81, Reversible and covalent binding of drugs tohuman serum albumin: methodological approaches and physiologicalrelevance.

The drugs and/or metabolites and/or small molecules bound to SA may ormay not overlap with the drugs and/or metabolites and/or small moleculeswhich do not substantially inhibit or compete with serum albumin forbinding to the single variable domain. The drugs and/or metabolitesinclude, but are preferably not limited to warfarin, ibuprofen, vitaminB6, theta bilirubin, hemin, thyroxine, fatty acids, acetaldehyde, fattyacid metabolites, acyl glucuronide, metabolites of bilirubin, halothane,salicylate, benzodapenes and 1-O-gemfibrozil-B-D-glucuronide. Thisinhibition or competition with serum albumin for binding to the singlevariable domain by small molecules may occur by both direct displacementand by allosteric effects as described for small molecule bindinginduced changes on the binding of other small molecules, see Ascenzi etal. (2006) Mini Rev. Med. Chem. 6(4):483-9. Allosteric modulation ofdrug binding to human serum albumin, and Ghuman J. et al. (2005) J. Mol.Biol. 353(1):38-52 Structural basis of the drug-binding to human serumalbumin. In one embodiment the small molecule, either alone, or inconcert with one or more other small molecules, and/or metabolites,and/or proteins and/or drugs, binds serum albumin. In anotherembodiment, the small molecule either alone, or in concert with one ormore other small molecules, and/or metabolites, and/or proteins and/ordrugs, does not substantially inhibit or compete with serum albumin forbinding to the single variable domain. In another embodiment, the smallmolecule, either alone or in concert with one or more other smallmolecules, and/or metabolites, and/or proteins and/or drugs,substantially inhibits or competes with serum albumin for binding to thesingle variable domain.

The single variable domain can be an antibody single variable domain.The antibody single variable domain can be a VH3 domain. The antibodysingle variable domain can be a V kappa domain. The single variabledomain can comprise one or more non-immunoglobulin scaffolds. Thenon-immunoglobulin scaffold can include one or more of, but ispreferably not limited to, CTLA-4, lipocallin, SpA, GroEL andfibronectin, and includes an Affibody™ and an Avimer™.

Described herein is a method of selecting a single variable domain whichbinds serum albumin, comprising selecting a first variable domain by itsability to bind to serum albumin in the presence of one or moremetabolites and/or drugs, where the selection is performed in thepresence of the one or more metabolites and/or drugs. Also describedherein is a method for producing a dual specific ligand comprising afirst immunoglobulin single variable domain having a first bindingspecificity for serum albumin in the presence of one or metaboliteand/or drug, and a second immunoglobulin single variable domain having asecond binding specificity, the method comprising the steps of: (a)selecting a first variable domain by its ability to bind to a firstepitope in the presence of one or more metabolites and/or drugs, (b)selecting a second variable domain by its ability to bind to a secondepitope, (c) combining the variable domains; and (d) selecting theligand by its ability to bind to serum albumin in the presence of saidone or more metabolites and/or ligands and said second epitopes. Thismethod can also include a step where the first variable domain isselected for binding to said first epitope in absence of a complementaryvariable domain, and/or where the first variable domain is selected forbinding to said first epitope in the presence of a third complementaryvariable domain in which said third variable domain is different fromsaid second variable domain. These selection steps can be performed inthe presence of a mixture of metabolites and/or drugs and/or proteinsand/or small molecules. The selection steps can also be performed asfollows: (a) selecting single variable domains which bind serum albuminin the presence of a first metabolite and/or drug and/or small molecule;and (b) from domains selected in step (a), a domain is selected in thepresence of a second metabolite and/or drug and/or small molecule. Alsoencompassed is a method for producing a dual specific ligand having afirst immunoglobulin single variable domain having a first bindingspecificity for serum albumin in the presence of one or metaboliteand/or drug and/or small molecule, and a second immunoglobulin singlevariable domain having a second binding specificity, the method havingthe steps of: (a) selecting first variable domains by their ability tobind to serum albumin in the presence of one or more metabolites and/ordrugs and/or small molecules, (b) selecting second variable domains bytheir ability to bind to an epitope, (c) combining the variable domainsto provide ligands comprising a first and a second variable domain; and(d) from the ligands provided by step (c), and selecting a ligand by itsability to bind to serum albumin in the presence of the one or moremetabolites and/or drugs and its ability to bind to said epitopes,thereby producing a dual specific ligand. In one embodiment, the firstvariable domain is selected for binding to serum albumin in absence of acomplementary variable domain. In another embodiment, the first variabledomain is selected for binding to the first epitope in the presence of acomplementary variable domain in which the complementary variable domainis different from the second variable domain.

Described herein is a ligand comprising a single variable domain, wherethe single variable domain specifically binds serum albumin in vitroboth at pH 7 and at an intracellular compartment pH, and where thesingle variable domain is a non-naturally occurring single variabledomain. Also described herein is a ligand comprising an antibody singlevariable domain, where the antibody single variable domain specificallybinds serum albumin in vitro both at pH 7 and at an intracellularcompartment pH. In one embodiment the intracellular compartment pHranges from 4.8 to 5.2. In another embodiment, the binding of serumalbumin to the antibody single variable domain does not substantiallyinhibit the binding of FcRn to the serum albumin, as determined by an invitro Surface Plasmon Resonance competition assay. In anotherembodiment, the antibody single variable domain is an antibody heavychain single variable domain. The antibody heavy chain single variabledomain can be a VH3 single variable domain, and the VH3 single variabledomain can be a human VH3 single variable domain, in additionalembodiments. In another embodiment, the antibody single variable domainis an antibody light chain single variable domain. The antibody lightchain single variable domain is a Vkappa single variable domain in oneembodiment, and in another embodiment is a Vlambda single variabledomain.

In another embodiment, the antibody single variable domain comprises oneor more of antibody CDR regions selected from the group consisting of:CDR1, CDR2 and CDR3. In another embodiment, the antibody single variabledomain comprises a scaffold selected from the group consisting of:CTLA-4, lipocallin, staphylococcal protein A (SpA), GroEL, GroES,transferrin and fibronectin. The binding of serum albumin to the singlevariable domain does not substantially compete with the binding of FcRnto serum albumin in one embodiment, and in another embodiment theantibody single variable domain specifically binds serum albumin with anequilibrium dissociation constant (Kd) of less than or equal to 300 nM.

In another embodiment, the antibody single variable domain furthercomprises at least one additional antibody single variable domain, wherethe additional antibody single variable domain specifically binds anantigen other than serum albumin at pH 7, but does not bind the antigenat the intracellular compartment pH. Also described herein is a methodof directing an antigen for degradation in an individual comprisingadministering a ligand comprising a single variable domain, such as anantibody single variable domain, which specifically binds serum albuminin vitro both at pH 7 and at an intracellular compartment pH, to theindividual, the ligand further comprising at least one additionalantibody single variable domain comprising a single variable domain,e.g., an antibody single variable domain, where the antigen other thanserum albumin is the antigen which is targeted for degradation.

In one embodiment of the ligands of the invention, the specific bindingof human serum albumin by the antibody single variable domain is notblocked by binding of a pre-determined drug and/or a metabolite and/or asmall molecule to one or more sites on the human serum albumin. In theseembodiments, the additional antibody single variable domain can be anantibody heavy chain single variable domain or an antibody light chainsingle variable domain which comprises one or more antibody CDRsselected from the group consisting of: CDR1, CDR2 and/or CDR3. Thesingle variable domains can comprises a scaffold selected from the groupconsisting of: CTLA-4, lipocallin, staphylococcal protein A (SpA),GroEL, GroES, transferrin and fibronectin.

Another embodiment of a ligand described herein, is a ligand whichcomprises a single variable domain, where the single variable domain isa non-naturally occurring single variable domain, where the singlevariable domain specifically binds human serum albumin in vitro both atpH 7 and at an intracellular compartment pH, where the specific bindingof human serum albumin by the single variable domain is not blocked bybinding of a pre-determined drug and/or a metabolite and/or a smallmolecule to one or more sites on the human serum albumin, where the oneor more sites on human serum albumin include Sudlow site 1 and Sudlowsite 2. or the one or more sites are located on one or more domains ofhuman serum albumin selected from the group consisting of: domain I,domain II and domain III.

Linkers

Connecting an AlbudAb™ (a dAb which specifically binds serum albumin)(anti-serum albumin domain antibody or single variable domain) toanother biologically active moiety can be obtained by recombinantengineering techniques. Basically, the genes encoding both proteins ofinterest are fused in frame. Several formats can be considered where theanti-serum albumin domain antibody is either at the N-terminal end ofthe fusion (i.e. AlbudAb™-Y where Y is a biologically activepolypeptide), at the C-terminal end of the fusion (i.e. Z-AlbudAb™ whereZ is a biologically active peptide). In some instances, one may considerfusing more than one biologically active polypeptide to an AlbudAb™ (adAb which specifically binds serum albumin), resulting in a number ofpossibilities regarding the fusion design. For example, the fusion couldbe as follows: Z—Y-AlbudAb™, Z-AlbudAb™-Y or AlbudAb™-Z-Y.

In all these fusion molecules, two polypeptides are covalently linkedtogether via at least one peptide bond. In its most simplistic approach,the AlbudAb™ (a dAb which specifically binds serum albumin) and thebiologically polypeptide(s) are directly linked. Thus, the junctionbetween the AlbudAb™ (a dAb which specifically binds serum albumin) andthe polypeptide would be as follows:

a) For an AlbudAb™ (a dAb which specifically binds serum albumin) at theC-terminal end,

Where the AlbudAb™ is a VK:—

xxxDIQxxxNIQxxxAIQxxxAIRxxxVIWxxxDIVxxxDVVxxxEIVxxxETT

Where the AlbudAb™ (a dAb which specifically binds serum albumin) is aVλ:—

xxxQSVxxxQSAxxxSYExxxSSExxxSYVxxxLPVxxxQPVxxxQLVxxxQAVxxxNFMxxxQTVxxxQAG

Where the AlbudAb™ (a dAb which specifically binds serum albumin) is aVH (e.g., human VH):—

xxxQVQxxxQMQxxxEVQxxxQITxxxQVTxxxQLQ

Where the AlbudAb™ (a dAb which specifically binds serum albumin) is aVHH (e.g., Camelid heavy chain variable domain):—

xxxEVQxxxQVQxxxDVQxxxQVKxxxAVQ

b) For an AlbudAb™ (a dAb which specifically binds serum albumin) at theN-terminal end,

Where the AlbudAb™ (a dAb which specifically binds serum albumin) is aVK:—

KVEIKxxx KLEIKxxx KVDIKxxx RLEIKxxx EIKRxxx

Where the AlbudAb™ (a dAb which specifically binds serum albumin) is aVλ:—

KVDVLxxx KLDVLxxx QLDVLxxx

Where the AlbudAb™ (a dAb which specifically binds serum albumin) is aVH (e.g., human VH):—

VTVSSxxx

Where the AlbudAb™ (a dAb which specifically binds serum albumin) is aVHH (e.g., Camelid heavy chain variable domain):—

VTVSSxxx

‘xxx’ represents the first or last three amino acids of the (first)biologically active polypeptide fused to the AlbudAb™ (a dAb whichspecifically binds serum albumin).

However, there may be instances where the production of a recombinantfusion protein that recovers the functional activities of bothpolypeptides may be facilitated by connecting the encoding genes with abridging DNA segment encoding a peptide linker that is spliced betweenthe polypeptides connected in tandem. Optimal peptide linker length isusually devised empirically: it can be as short as one amino acid orextend up to 50 amino acids. Linkers of different designs have beenproposed and are well know in the art. The following examples are meantto provide a broad—but not comprehensive—list of possible linkerapproaches:

1. Flexible Linkers:

Flexible linkers are designed to adopt no stable secondary structurewhen connecting two polypeptide moieties, thus allowing a range ofconformations in the fusion protein. These linkers are preferablyhydrophilic in nature to prevent these from interacting with one or bothfused polypeptides. Usually small polar residues such as glycine andserine are prevalent in those linkers in order to increase the flexibleand hydrophilic characteristics of the peptide backbone, respectively.The length of these linkers is variable and best determined eitherempirically or with the aid of 3D computing approaches. In general, apreferred linker length will be the smallest compatible with goodexpression, good solubility and full recovery of the native functionsand structures of interest. Because of their flexible characteristics,flexible linkers may constitute good substrates for endogenousproteases. In general, unless it is a desirable feature flexible linkersare devoid of amino acids such as charged amino acids or largehydrophobic/aromatic which are readily recognized by endogenousproteases with broad substrate specificity. In addition cysteineresidues are preferably avoided since free cysteines can react togetherto form cysteines, thereby resulting in (i) bridging two fusion proteinsvia the linkers, and/or (ii) compromised expression/folding of thefusion protein if one or more of the bioactive polypeptides comprisesone or more cysteine residue (‘cysteine scrambling’).

Examples of flexible linkers are: (i) glycine-rich linkers based on therepetition of a (GGGGS)_(y) motif where y is at least 1, though y can be2, 3, 4, 5, 6, 7, 8 and 9, or more (see PCT International PublicationsNo: EP 0 753 551, U.S. Pat. No. 5,258,498, EP 0 623 679), (ii)serine-rich linkers based on the repetition of a (SSSSG)_(y) motif wherey is at least 1, though y can be 2, 3, 4, 5, 6, 7, 8 and 9, or more (seePCT International Publications No: EP 0 573 551, U.S. Pat. No.5,525,491).

2. Constrained Linkers:

Constrained linkers are designed to adopt a stable secondary structurewhen connecting two polypeptide moieties, thus restricting the range ofconformations in the fusion protein. Such linkers usually adopt ahelical structure spanning several turns. Again the length of theselinkers is variable and best determined either empirically or with theaid of computing approaches. The main reason for choosing constrainedlinkers is to maintain the longest distance between each polypeptide ofthe fusion. This is particularly relevant when both polypeptides have atendency to form hetero-aggregates. By virtue of their structure,constrained linkers can also be more resistant to proteolyticdegradation, thereby offering an advantage when injected in vivo.

Examples of constrained linkers are cited in PCT InternationalPublications No: WO 00/24884 (e.g. SSSASASSA, GSPGSPG, or ATTTGSSPGPT),U.S. Pat. No. 6,132,992 (e.g. helical peptide linkers).

3. ‘Natural’ Linkers:

Natural linkers are polypeptide sequences (of variable lengths) that—byopposition—are not synthetic, i.e. the polypeptide sequences composingthe linkers are found in nature. Natural linkers can be either flexibleor constrained and can be very diverse in amino acid sequence andcomposition. Their degree of resistance to proteolysis depends on whichproteins they originate from and which biological environment theseproteins are facing in nature (extracellular, intracellular,prokaryotic, eukaryotic, etc). One class of linkers is particularlyrelevant for the development of biological therapeutics in man: linkersbased on peptides found in human proteins. Indeed such linkers are bynature non—or very weakly—immunogenic and therefore potentially saferfor human therapy.

Examples of natural linkers are: (i) KESGSVSSEQLAQFRSLD (see Bird et al.(1988) Science, 242, 423-426), (ii) sequences corresponding to the hingedomain of immunoglobulins devoid of light chains (see Hamers-Castermanet al. (1993) Nature, 363, 446-448 and PCT International Publication No:WO 096/34103). Examples of linkers for use with anti-albumin domainantibodies (e.g., human, humanized, camelized human or Camelid VHHdomain antibodies) are EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP andGTNEVCKCPKCP. Other linkers derived from human and camelid hinges aredisclosed in EP0656946, incorporated herein by reference. The hingederived linkers can have variable lengths, for example from 0 to about50 amino acids, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49amino acids.

As used herein, “drug” refers to any compound (e.g., small organicmolecule, nucleic acid, polypeptide) that can be administered to anindividual to produce a beneficial, therapeutic or diagnostic effectthrough binding to and/or altering the function of a biological targetmolecule in the individual. The target molecule can be an endogenoustarget molecule encoded by the individual's genome (e.g. an enzyme,receptor, growth factor, cytokine encoded by the individual's genome) oran exogenous target molecule encoded by the genome of a pathogen (e. g.an enzyme encoded by the genome of a virus, bacterium, fungus, nematodeor other pathogen).

The drug composition can be a conjugate wherein the drug is covalentlyor noncovalently bonded to the polypeptide binding moiety. The drug canbe; covalently or noncovalently bonded to the polypeptide binding moietydirectly or indirectly (e.g., through a suitable linker and/ornoncovalent binding of complementary binding partners (e.g., biotin andavidin)). When complementary binding partners are employed, one of thebinding partners can be covalently bonded to the drug directly orthrough a suitable linker moiety, and the complementary binding partnercan be covalently bonded to the polypeptide binding moiety directly orthrough a suitable linker moiety. When the drug is a polypeptide orpeptide, the drug composition can be a fusion protein, wherein thepolypeptide or peptide, drug and the polypeptide binding moiety arediscrete parts (moieties) of a continuous polypeptide chain. Asdescribed herein, the polypeptide binding moieties and polypeptide drugmoieties can be directly bonded to each other through a peptide bond, orlinked through a suitable amino acid, or peptide or polypeptide linker.

Decreased Immunogenicity

Described herein is a method of reducing the immunogenicity of apharmaceutical agent, comprising modifying said agent so that the agentfurther contains a single variable domain region, where the singlevariable domain specifically binds serum albumin in vivo and/or ex vivo,and where the agent can include a drug, a metabolite, a ligand, anantigen and a protein. The serum albumin can be human serum albumin. Thesingle variable domain can be an immunoglobulin single variable domain.The immunoglobulin single variable domain can be a VH antibody singlevariable domain. The VH single variable domain can be a VH3 singlevariable domain. The VH3 single variable domain can be a human VH3single variable domain. The single variable domain can be a Vkappa or aVlambda antibody single variable domain. The antibody single variabledomain can comprise a set of four Kabat framework regions (FRs which areencoded by VH3 framework germ line antibody gene segments. The V_(H)3framework is selected from the group consisting of DP47, DP38 and DP45.The antibody single variable domain can contain a set of four Kabatframework regions (FRs), which are encoded by VKappa framework germ lineantibody gene segments. A nonlimiting example of a Kappa framework isDPK9. The single variable domain can contain an immunoglobulin ornon-immunoglobulin scaffold which contains CDR1, CDR2 and/or CDR3regions, wherein at least one of the CDR1, CDR2 and CDR3 regions is froman antibody variable domain which specifically binds serum albumin. Thenon-immunoglobulin scaffold can include, but is preferably not limitedto, CTLA-4, lipocallin, SpA, Affibody™, GroEL, Avimers™ and fibronectin.The serum albumin can be human serum albumin. The immunoglobulin singlevariable domain and/or the non-immunoglobulin single variable domain canspecifically bind to human serum albumin with a Kd of less than 300 nM.The immunoglobulin single variable domain and/or the non-immunoglobulinsingle variable domain can specifically bind to both human serum albuminand one or more non-human serum albumins, with Kd values within 10 foldof each other. The immunoglobulin single variable domain and/ornon-immunoglobulin single variable domain can specifically bind to bothhuman serum albumin and one or more non-human serum albumins, andwherein the T beta half life of the ligand is substantially the same asthe T beta half life of human serum albumin in a human host. Further,the immunoglobulin single variable domain and/or non-immunoglobulinsingle variable domain can specifically bind to Domain II of human serumalbumin. The immunoglobulin single variable domain and/or thenon-immunoglobulin single variable domain can further specifically bindserum albumin both at a natural serum pH, and at an intracellularvesicle pH. The specific binding of serum albumin by said immunoglobulinsingle variable domain and/or the non-immunoglobulin single variabledomain is preferably not substantially blocked by binding of drugsand/or metabolites to one or more sites on serum albumin. In oneembodiment, the specific binding of serum albumin by the single variabledomain does not alter the binding characteristics of serum albumin fordrugs and/or metabolites and/or small molecules bound to SA. In oneembodiment the method of modifying the agent results in the formation ofan modified agent having a formula comprising: a-(X)n1-b-(Y)n2-c-(Z)n3-dor a-(Z)n3-b-(Y)n2-c-(X)n-d, wherein X is a polypeptide drug that hasbinding specificity for a first target; Y is a single variable domain,e.g. an antibody single variable domain that specifically binds serumalbumin in vivo and/or ex vivo; Z is a polypeptide drug that has bindingspecificity for a second target; a, b, c and d are independently apolypeptide comprising one to about amino acid residues or absent; n1 isone to about 10; n2 is one to about 10; and n3 is zero to about 10. In afurther embodiment, when n1 and n2 are both one and n3 is zero, X doesnot comprise an antibody chain or a fragment of an antibody chain.

Described herein is a method of reducing the immunogenicity of apharmaceutical agent, comprising modifying the agent so that the agentfurther comprises a single variable domain, where the single variabledomain specifically binds serum albumin, where the single variabledomain is a non-naturally occurring single variable domain, and wherethe agent is selected from the group consisting of: a drug, ametabolite, a ligand, an antigen and a protein. Also described herein isa method of reducing the immunogenicity of a pharmaceutical agent,comprising modifying the agent so that the agent further comprises anantibody single variable domain, where the antibody single variabledomain specifically binds serum albumin, and where the agent is selectedfrom the group consisting of: a drug, a metabolite, a ligand, an antigenand a protein. In one embodiment, the antibody single variable domain isan antibody heavy chain single variable domain, e.g., antibody VH3single variable domain, or a human antibody VH3 single variable domain.In another embodiment, the antibody single variable domain is anantibody light chain single variable domain, e.g., an antibody Vkappa oran antibody Vlambda single variable domain. In one embodiment, theantibody single variable domain comprises CDR1, CDR2 and CDR3 regions,where at least one of the CDR1, CDR2 and CDR3 regions is from anantibody variable domain which specifically binds serum albumin, andoptionally further comprises a scaffold selected from the groupconsisting of: CTLA-4, lipocallin, staphylococcal protein A (SpA),GroEL, GroES, transferrin and fibronectin. In another embodiment ofthese methods, the single variable domain, e.g., the antibody singlevariable domain specifically binds to human serum albumin with a kd ofless than 300 nM, and in another embodiment of these methods, the singlevariable domain, e.g., the antibody single variable domain, specificallybinds to human serum albumin and one or more non-human serum albumins,with Kd values within 10 fold of each other. In another embodiment ofthese methods, the single variable domain, e.g., the antibody singlevariable domain, specifically binds to human serum albumin and anon-human serum albumin, and the T beta half life of the ligand issubstantially the same as the T beta half life of human serum albumin ina human host. In another embodiment of these methods, the singlevariable domain, e.g., the antibody single variable domain, specificallybinds to Domain II of human serum albumin. In another embodiment ofthese methods, the single variable domain, e.g., the antibody singlevariable domain, specifically binds serum albumin both at a pH 7, and atan intracellular compartment pH.

The invention is further described, for the purposes of illustrationonly, in the following examples. As used herein, for the purposes of dAbnomenclature, human TNFα is referred to as TAR1 and human TNFα receptor1 (p55 receptor) is referred to as TAR2.

Example 1. Selection of a Dual Specific scFv Antibody (K8) DirectedAgainst Human Serum Albumin (HSA) and β-Galactosidase (β-Gal)

This example explains a method for making a dual specific antibodydirected against β-gal and HSA in which a repertoire of V_(κ) variabledomains linked to a germ line (dummy) V_(H) domain is selected forbinding to 3-gal and a repertoire of V_(H) variable domains linked to a-germ line (dummy) V_(κ) domain is selected for binding to HSA. Theselected variable V_(H) HSA and V_(κ) β-gal domains are then combinedand the antibodies selected for binding to β-gal and HSA. HSA is ahalf-life increasing protein found in human blood.

Four human phage antibody libraries were used in this experiment.

Library 1 -Germ line V_(κ)/DVT V_(H) 8.46 × 10⁷ Library 2 -Germ lineV_(κ)/NNK V_(H) 9.64 × 10⁷ Library 3 -Germ line V_(H)/DVT V_(κ) 1.47 ×10⁸ Library 4 -Germ line V_(H)/NNK V_(κ) 1.45 × 10⁸

All libraries are based on a single human framework for V_(H)(V3-23/DP47 and JH4b) and V_(κ) (O12/O2/DPK9 and J_(κ)1) with side chaindiversity incorporated in complementarity determining regions (CDR2 andCDR3).

Library 1 and Library 2 contain a dummy V_(κ) sequence, whereas thesequence of V_(H) is diversified at positions H50, H52, H52a, H53, H55,H56, H58, H95, H96, H97 and H98 (DVT or NNK encoded, respectively) (FIG.1). Library 3 and Library 4 contain a dummy V_(H) sequence, whereas thesequence of V_(κ) is diversified at positions L50, L53, L91, L92, L93,L94 and L96 (DVT or NNK encoded, respectively) (FIG. 1). The librariesare in phagemid pIT2/ScFv format (FIG. 2) and have been preselected forbinding to generic ligands, Protein A and Protein L, so that themajority of clones in the unselected libraries are functional. The sizesof the libraries shown above correspond to the sizes after preselection.Library 1 and Library 2 were mixed prior to selections on antigen toyield a single V_(H)/dummy V_(κ) library and Library 3 and Library 4were mixed to form a single V_(κ)/dummy V_(H) library.

Three rounds of selections were performed on β-gal using V_(κ)/dummyV_(H) library and three rounds of selections were performed on HSA usingV_(H)/dummy V_(κ) library. In the case of β-gal the phage titres went upfrom 1.1×10⁶ in the first round to 2.0×10⁸ in the third round. In thecase of HSA the phage titres went up from 2×10⁴ in the first round to1.4×10⁹ in the third round. The selections were performed as describedby Griffith et al., (1993), except that KM13 helper phage (whichcontains a pIII protein with a protease cleavage site between the D2 andD3 domains) was used and phage were eluted with 1 mg/ml trypsin in PBS.The addition of trypsin cleaves the pIII proteins derived from thehelper phage (but not those from the phagemid) and elutes boundscFv-phage fusions by cleavage in the c-myc tag (FIG. 2), therebyproviding a further enrichment for phages expressing functional scFvsand a corresponding reduction in background (Kristensen & Winter,Folding & Design 3: 321-328, Jul. 9, 1998). Selections were performedusing immunotubes coated with either HSA or β-gal at 100 μg/mlconcentration.

To check for binding, 24 colonies from the third round of each selectionwere screened by monoclonal phage ELISA. Phage particles were producedas described by Harrison et al., Methods Enzymol. 1996; 267:83-109.96-well ELISA plates were coated with 100 μl of HSA or β-gal at 10 μg/mlconcentration in PBS overnight at 4° C. A standard ELISA protocol wasfollowed (Hoogenboom et al., 1991) using detection of bound phage withanti-M13-HRP conjugate. A selection of clones gave ELISA signals ofgreater than 1.0 with 50 μl supernatant.

Next, DNA preps were made from V_(H)/dummy V_(κ) library selected on HSAand from V_(κ)/dummy V_(H) library selected on β-gal using the QIAprepSpin Miniprep kit (Qiagen). To access most of the diversity, DNA prepswere made from each of the three rounds of selections and then pulledtogether for each of the antigens. DNA preps were then digested withSalI/NotI overnight at 37° C. Following gel purification of thefragments, V_(κ) chains from the V_(κ)/dummy V_(H) library selected onβ-gal were ligated in place of a dummy V_(κ) chain of the V_(H)/dummyV_(κ) library selected on HSA creating a library of 3.3×10⁹ clones.

This library was then either selected on HSA (first round) and β-gal(second round), HSA/β-gal selection, or on β-gal (first round) and HSA(second round), β-gal/HSA selection. Selections were performed asdescribed above. In each case after the second round 48 clones weretested for binding to HSA and β-gal by the monoclonal phage ELISA (asdescribed above) and by ELISA of the soluble scFv fragments. Solubleantibody fragments were produced as described by Harrison et al.,(1996), and standard ELISA protocol was followed Hoogenboom et al.(1991) Nucleic Acids Res., 19: 4133, except that 2% Tween/PBS was usedas a blocking buffer and bound scFvs were detected with Protein L-HRP.Three clones (E4, E5 and E8) from the HSA/β-gal selection and two clones(K8 and K10) from the β-gal/HSA selection were able to bind bothantigens. scFvs from these clones were PCR amplified and sequenced asdescribed by Ignatovich et al., (1999) J Mol Biol 1999 Nov. 26;294(2):457-65, using the primers LMB3 and pHENseq. Sequence analysisrevealed that all clones were identical. Therefore, only one cloneencoding a dual specific antibody (K8) was chosen for further work (FIG.3).

Example 2. Characterisation of the Binding Properties of the K8 Antibody

Firstly, the binding properties of the K8 antibody were characterised bythe monoclonal phage ELISA. A 96-well plate was coated with 100 μl ofHSA and β-gal alongside with alkaline phosphatase (APS), bovine serumalbumin (BSA), peanut agglutinin, lysozyme and cytochrome c (to checkfor cross-reactivity) at 10 μg/ml concentration in PBS overnight at 4°C. The phagemid from K8 clone was rescued with KM13 as described byHarrison et al., (1996) and the supernatant (50 μl) containing phageassayed directly. A standard ELISA protocol was followed (Hoogenboom etal., 1991) using detection of bound phage with anti-M13-HRP conjugate.The dual specific K8 antibody was found to bind to HSA and β-gal whendisplayed on the surface of the phage with absorbance signals greaterthan 1.0 (FIG. 4). Strong binding to BSA was also observed (FIG. 4).Since HSA and BSA are 76% homologous on the amino acid level, it is notsurprising that K8 antibody recognised both of these structurallyrelated proteins. No cross-reactivity with other proteins was detected(FIG. 4).

Secondly, the binding properties of the K8 antibody were tested in asoluble scFv ELISA. Production of the soluble scFv fragment was inducedby IPTG as described by Harrison et al., (1996). To determine theexpression levels of K8 scFv, the soluble antibody fragments werepurified from the supernatant of 50 ml inductions using ProteinA-Sepharose columns as described by Harlow and Lane, Antibodies: aLaboratory Manual, (1988) Cold Spring Harbor. OD₂₈₀ was then measuredand the protein concentration calculated as described by Sambrook etal., (1989). K8 scFv was produced in supernatant at 19 mg/l.

A soluble scFv ELISA was then performed using known concentrations ofthe K8 antibody fragment. A 96-well plate was coated with 100 μl of HSA,BSA and β-gal at 10 μg/ml and 100 μl of Protein A at 1 μg/mlconcentration. 50 μl of the serial dilutions of the K8 scFv was appliedand the bound antibody fragments were detected with Protein L-HRP. ELISAresults confirmed the dual specific nature of the K8 antibody (FIG. 5).

To confirm that binding to β-gal is determined by the V_(κ) domain andbinding to HSA/BSA by the V_(H) domain of the K8 scFv antibody, theV_(κ) domain was cut out from K8 scFv DNA by SalI/NotI digestion andligated into a SalI/NotI digested pIT2 vector containing dummy V_(H)chain (FIGS. 1 and 2). Binding characteristics of the resulting cloneK8V_(κ)/dummy V_(H) were analysed by soluble scFv ELISA. Production ofthe soluble scFv fragments was induced by IPTG as described by Harrisonet al., (1996) and the supernatant (50μ) containing scFvs assayeddirectly. Soluble scFv ELISA was performed as described in Example 1 andthe bound scFvs were detected with Protein L-HRP. The ELISA resultsrevealed that this clone was still able to bind β-gal, whereas bindingto BSA was abolished (FIG. 6).

Example 3. Selection of Single V_(H) Domain Antibodies Antigens A and Band Single V_(κ) Domain Antibodies Directed Against Antigens C and D

This example describes a method for making single V_(H) domainantibodies directed against antigens A and B and single V_(κ) domainantibodies directed against antigens C and D by selecting repertoires ofvirgin single antibody variable domains for binding to these antigens inthe absence of the complementary variable domains.

Selections and characterisation of the binding clones is performed asdescribed previously (see Example 5, PCT/GB 02/003014). Four clones arechosen for further work:

VH1—Anti A V_(H) VH2—Anti B V_(H) VK1—Anti C V_(κ) VK2—Anti D V_(κ)

The procedures described above in Examples 1-3 may be used, in a similarmanner as that described, to produce dimer molecules comprisingcombinations of V_(H) domains (i.e., V_(H)-V_(H) and combinations ofV_(L) domains (V_(L)-V_(L) ligands).

Example 4. Creation and Characterisation of the Dual Specific ScFvAntibodies (VH1/VH2 Directed Against Antigens A and B and VK1/VK2Directed Against Antigens C and D)

This example demonstrates that dual specific ScFv antibodies(V_(H)1/V_(H)2 directed against antigens A and B and VK1/VK2 directedagainst antigens C and D) could be created by combining V_(κ) and V_(H)single domains selected against respective antigens in a ScFv vector.

To create dual specific antibody VH1/VH2, VH1 single domain is excisedfrom variable domain vector 1 (FIG. 7) by NcoI/XhoI digestion andligated into NcoI/XhoI digested variable domain vector 2 (FIG. 7) tocreate VH1/variable domain vector 2. VH2 single domain is PCR amplifiedfrom variable domain vector 1 using primers to introduce SalIrestriction site to the 5′ end and Nod restriction site to the 3′ end.The PCR product is then digested with SalI/NotI and ligated intoSalI/NotI digested VH1/variable domain vector 2 to createVH1/VH2/variable domain vector 2.

VK1/VK2/variable domain vector 2 is created in a similar way. The dualspecific nature of the produced VH1/VH2 ScFv and VK1/VK2 ScFv is testedin a soluble ScFv ELISA as described previously (see Example 6, PCT/GB02/003014). Competition ELISA is performed as described previously (seeExample 8, PCT/GB 02/003014).

Possible Outcomes:

-   -   VH1/VH2 ScFv is able to bind antigens A and B simultaneously    -   VK1/VK2 ScFv is able to bind antigens C and D simultaneously    -   VH1/VH2 ScFv binding is competitive (when bound to antigen A,        VH1/VH2 ScFv cannot bind to antigen B)    -   VK1/VK2 ScFv binding is competitive (when bound to antigen C,        VK1/VK2 ScFv cannot bind to antigen D)

Example 5. Construction of Dual Specific VH1/VH2 Fab and VK1/VK2 Fab andAnalysis of their Binding Properties

To create VH1/VH2 Fab, VH1 single domain is ligated into NcoI/XhoIdigested CH vector (FIG. 8) to create VH1/CH and VH2 single domain isligated into SalI/NotI digested CK vector (FIG. 9) to create VH2/CK.Plasmid DNA from VH1/CH and VH2/CK is used to co-transform competent E.coli cells as described previously (see Example 8, PCT/GB02/003014).

The clone containing VH1/CH and VH2/CK plasmids is then induced by IPTGto produce soluble VH1/VH2 Fab as described previously (see Example 8,PCT/GB 02/003014).

VK1/VK2 Fab is produced in a similar way.

Binding properties of the produced Fabs are tested by competition ELISAas described previously (see Example 8, PCT/GB 02/003014).

Possible Outcomes:

-   -   VH1/VH2 Fab is able to bind antigens A and B simultaneously    -   VK1/VK2 Fab is able to bind antigens C and D simultaneously    -   VH1/VH2 Fab binding is competitive (when bound to antigen A,        VH1/VH2 Fab cannot bind to antigen B)    -   VK1/VK2 Fab binding is competitive (when bound to antigen C,        VK1/VK2 Fab cannot bind to antigen D)

Example 6 Chelating dAb Dimers Summary

VH and VK homo-dimers are created in a dAb-linker-dAb format usingflexible polypeptide linkers. Vectors were created in the dAb linker-dAbformat containing glycine-serine linkers of different lengths3U:(Gly₄Ser)₃, 5U:(Gly₄Ser)₅, 7U:(Gly₄Ser)₇. Dimer libraries werecreated using guiding dAbs upstream of the linker: TAR1-5 (VK),TAR1-27(VK), TAR2-5(VH) or TAR2-6(VK) and a library of correspondingsecond dAbs after the linker. Using this method, novel dimeric dAbs wereselected. The effect of dimerisation on antigen binding was determinedby ELISA and Biacore studies and in cell neutralisation and receptorbinding assays. Dimerisation of both TAR1-5 and TAR1-27 resulted insignificant improvement in binding affinity and neutralisation levels.

1.0 Methods 1.1 Library Generation 1.1.1 Vectors

pEDA3U, pEDA5U and pEDA7U vectors were designed to introduce differentlinker lengths compatible with the dAb-linker-dAb format. For pEDA3U,sense and anti-sense 73-base pair oligo linkers were annealed using aslow annealing program (95° C.-5 mins, 80° C.-10 mins, 70° C.-15 mins,56° C.-15 mins, 42° C. until use) in buffer containing 0.1MNaCl, 10 mMTris-HCl pH7.4 and cloned using the Xho1 and Not1 restriction sites. Thelinkers encompassed 3 (Gly₄Ser) units and a stuffer region housedbetween SalI and Not1 cloning sites (scheme 1). In order to reduce thepossibility of monomeric dAbs being selected for by phage display, thestuffer region was designed to include 3 stop codons, a Sac1 restrictionsite and a frame shift mutation to put the region out of frame when nosecond dAb was present. For pEDA5U and 7U due to the length of thelinkers required, overlapping oligo-linkers were designed for eachvector, annealed and elongated using Klenow. The fragment was thenpurified and digested using the appropriate enzymes before cloning usingthe Xho1 and Not1 restriction sites.

1.1.2 Library Preparation

The N-terminal V gene corresponding to the guiding dAb was clonedupstream of the linker using Nco1 and Xho1 restriction sites. VH geneshave existing compatible sites, however cloning VK genes required theintroduction of suitable restriction sites. This was achieved by usingmodifying PCR primers (VK-DLIBF: 5′ cggccatggcgtcaacggacat; VKXho1R: 5′atgtgcgctcgagcgtttgattt 3′) in 30 cycles of PCR amplification using a2:1 mixture of SuperTaq (HTBiotechnology Ltd) and pfu turbo(Stratagene). This maintained the Nco1 site at the 5′ end whiledestroying the adjacent Sal1 site and introduced the Xho1 site at the 3′end. 5 guiding dAbs were cloned into each of the 3 dimer vectors: TAR1-5(VK), TAR1-27(VK), TAR2-5(VH), TAR2-6(VK) and TAR2-7(VK). All constructswere verified by sequence analysis.

Having cloned the guiding dAbs upstream of the linker in each of thevectors (pEDA3U, 5U and 7U): TAR1-5 (VK), TAR1-27(VK), TAR2-5(VH) orTAR2-6(VK) a library of corresponding second dAbs were cloned after thelinker. To achieve this, the complimentary dAb libraries were PCRamplified from phage recovered from round 1 selections of either a VKlibrary against Human TNFα (at approximately 1×10⁶ diversity afterround 1) when TAR1-5 or TAR1-27 are the guiding dAbs, or a VH or VKlibrary against human p55 TNF receptor (both at approximately 1×10⁵diversity after round 1) when TAR2-5 or TAR2-6 respectively are theguiding dAbs. For VK libraries PCR amplification was conducted usingprimers in 30 cycles of PCR amplification using a 2:1 mixture ofSuperTaq and pfu turbo. VH libraries were PCR amplified using primers inorder to introduce a Sal1 restriction site at the 5′ end of the gene.The dAb library PCRs were digested with the appropriate restrictionenzymes, ligated into the corresponding vectors down stream of thelinker, using Sal1/Not1 restriction sites and electroporated intofreshly prepared competent TG1 cells.

The titres achieved for each library are as follows:

TAR1-5: pEDA3U=4×10⁸, pEDA5U=8×10⁷, pEDA7U=1×10⁸TAR1-27: pEDA3U=6.2×10⁸, pEDA5U=1×10⁸, pEDA7U=1×10⁹TAR2h-5: pEDA3U=4×10⁷, pEDA5U=2×10⁸, pEDA7U=8×10⁷TAR2h-6: pEDA3U=7.4×10⁸, pEDA5U=1.2×10⁸, pEDA7U=2.2×10⁸

1.2 Selections 1.2.1 TNFα

Selections were conducted using human TNFα passively coated onimmunotubes. Briefly, Immunotubes are coated overnight with 1-4 mls ofthe required antigen. The immunotubes were then washed 3 times with PBSand blocked with 2% milk powder in PBS for 1-2 hrs and washed a further3 times with PBS. The phage solution is diluted in 2% milk powder in PBSand incubated at room temperature for 2 hrs. The tubes are then washedwith PBS and the phage eluted with 1 mg/ml trypsin-PBS. Three selectionstrategies were investigated for the TAR1-5 dimer libraries. The firstround selections were carried out in immunotubes using human TNFα coatedat 1 μg/ml or 20 μg/ml with 20 washes in PBS 0.1% Tween. TG1 cells areinfected with the eluted phage and the titres are determined (e.g.,Marks et al J Mol Biol. 1991 Dec. 5; 222(3):581-97, Richmann et alBiochemistry. 1993 Aug. 31; 32(34):8848-55).

The titres recovered were:

pEDA3U=2.8×10⁷ (1 μg/ml TNF) 1.5×10⁸ (20 μg/ml TNF),pEDA5U=1.8×10⁷ (1 μg/ml TNF), 1.6×10⁸ (20 μg/ml TNF)pEDA7U=8×10⁶ (1 μg/ml TNF), 7×10⁷ (20 μg/ml TNF).

The second round selections were carried out using 3 different methods.

1. In immunotubes, 20 washes with overnight incubation followed by afurther 10 washes.2. In immunotubes, 20 washes followed by 1 hr incubation at RT in washbuffer with (1 μg/ml TNFα) and 10 further washes.3. Selection on streptavidin beads using 33 pmoles biotinylated humanTNFα (Henderikx et al., 2002, Selection of antibodies againstbiotinylated antigens. Antibody Phage Display: Methods and protocols,Ed. O'Brien and Atkin, Humana Press). Single clones from round 2selections were picked into 96 well plates and crude supernatant prepswere made in 2 ml 96 well plate format.

Round 1 Human TNFαimmunotube Round 2 Round 2 Round 2 coating selectionselection selection concentration method 1 method 2 method 3 pEDA3U  1μg/ml 1 × 10⁹ 1.8 × 10⁹ 2.4 × 10¹⁰ pEDA3U 20 μg/ml 6 × 10⁹  1.8 × 10¹⁰8.5 × 10¹⁰ pEDA5U  1 μg/ml 9 × 10⁸ 1.4 × 10⁹ 2.8 × 10¹⁰ pEDA5U 20 μg/ml9.5 × 10⁹   8.5 × 10⁹ 2.8 × 10¹⁰ pEDA7U  1 μg/ml 7.8 × 10⁸   1.6 × 10⁸  4 × 10¹⁰ pEDA7U 20 μg/ml  1 × 10¹⁰   8 × 10⁹ 1.5 × 10¹⁰

For TAR1-27, selections were carried out as described previously withthe following modifications. The first round selections were carried outin immunotubes using human TNFα coated at 1 μg/ml or 20 μg/ml with 20washes in PBS 0.1% Tween. The second round selections were carried outin immunotubes using 20 washes with overnight incubation followed by afurther 20 washes. Single clones from round 2 selections were pickedinto 96 well plates and crude supernatant preps were made in 2 ml 96well plate format.

TAR1-27 titres are as follows:

Human TNFαimmunotube coating conc Round 1 Round 2 pEDA3U  1 μg/ml   4 ×10⁹   6 × 10⁹ pEDA3U 20 μg/ml   5 × 10⁹ 4.4 × 10¹⁰ pEDA5U  1 μg/ml 1.5 ×10⁹ 1.9 × 10¹⁰ pEDA5U 20 μg/ml 3.4 × 10⁹ 3.5 × 10¹⁰ pEDA7U  1 μg/ml 2.6× 10⁹   5 × 10⁹ pEDA7U 20 μg/ml   7 × 10⁹ 1.4 × 10¹⁰

1.2.2 TNF Receptor 1 (P55 Receptor; TAR2)

Selections were conducted as described previously for the TAR2h-5libraries only. Three rounds of selections were carried out inimmunotubes using either 1 μg/ml human p55 TNF receptor or 10 μg/mlhuman p55 TNF receptor with 20 washes in PBS 0.1% Tween with overnightincubation followed by a further 20 washes. Single clones from round 2and 3 selections were picked into 96 well plates and crude supernatantpreps were made in 2 ml, 96 well plate format.

TAR2h-5 titres are as follows:

Round 1 human p55 TNF receptor immunotube coating concentration Round 1Round 2 Round 3 pEDA3U  1 μg/ml 2.4 × 10⁶ 1.2 × 10⁷ 1.9 × 10⁹ pEDA3U 10μg/ml 3.1 × 10⁷   7 × 10⁷   1 × 10⁹ pEDA5U  1 μg/ml 2.5 × 10⁶ 1.1 × 10⁷5.7 × 10⁸ pEDA5U 10 μg/ml 3.7 × 10⁷ 2.3 × 10⁸ 2.9 × 10⁹ pEDA7U  1 μg/ml1.3 × 10⁶ 1.3 × 10⁷ 1.4 × 10⁹ pEDA7U 10 μg/ml 1.6 × 10⁷ 1.9 × 10⁷   3 ×10¹⁰

1.3 Screening

Single clones from round 2 or 3 selections were picked from each of the3U, 5U and 7U libraries from the different selections methods, whereappropriate. Clones were grown in 2×TY with 100 μg/ml ampicillin and 1%glucose overnight at 37° C. A 1/100 dilution of this culture wasinoculated into 2 mls of 2×TY with 100 μg/ml ampicillin and 0.1% glucosein 2 ml, 96 well plate format and grown at 37° C. shaking until OD600was approximately 0.9. The culture was then induced with 1 mM IPTGovernight at 30° C. The supernatants were clarified by centrifugation at4000 rpm for 15 mins in a sorval plate centrifuge. The supernatant prepsthe used for initial screening.

1.3.1 ELISA

Binding activity of dimeric recombinant proteins was compared to monomerby Protein A/L ELISA or by antigen ELISA. Briefly, a 96 well plate iscoated with antigen or Protein A/L overnight at 4° C. The plate washedwith 0.05% Tween-PBS, blocked for 2 hrs with 2% Tween-PBS. The sample isadded to the plate incubated for 1 hr at room temperature. The plate iswashed and incubated with the secondary reagent for 1 hr at roomtemperature. The plate is washed and developed with TMB substrate.Protein A/L-HRP or India-HRP was used as a secondary reagent. Forantigen ELISAs, the antigen concentrations used were 1 μg/ml in PBS forHuman TNFα and human THF receptor 1. Due to the presence of the guidingdAb in most cases dimers gave a positive ELISA signal, therefore offrate determination was examined by Biacore.

1.3.2 Biacore

Biacore analysis was conducted for TAR1-5 and TAR2h-5 clones. Forscreening, Human TNFα was coupled to a CM5 chip at high density(approximately 10000 RUs). 50 μl of Human TNFα(50 μg/ml) was coupled tothe chip at 5 μl/min in acetate buffer—pH5.5. Regeneration of the chipfollowing analysis using the standard methods is not possible due to theinstability of Human TNFα, therefore after each sample was analysed, thechip was washed for 10 mins with buffer.

For TAR1-5, clones supernatants from the round 2 selection were screenedby Biacore. 48 clones were screened from each of the 3U, 5U and 7Ulibraries obtained using the following selection methods:

R1: 1 μg/ml human TNFα immunotube, R2 1 μg/ml human TNFα immunotube,overnight wash.R1: 20 μg/ml human TNFα immunotube, R2 20 μg/ml human TNFα immunotube,overnight wash.R1: 1 μg/ml human TNFα immunotube, R2 33 pmoles biotinylated human TNFαon beads.R1: 20 μg/ml human TNFα immunotube, R2 33 pmoles biotinylated human TNFαbeads.

For screening, human p55 TNF receptor was coupled to a CM5 chip at highdensity (approximately 4000 RUs). 100 μl of human p55 TNF receptor (10μg/ml) was coupled to the chip at 5 μl/min in acetate buffer—pH5.5.Standard regeneration conditions were examined (glycine pH2 or pH3) butin each case antigen was removed from the surface of the chip thereforeas with TNFα, therefore after each sample was analysed, the chip waswashed for 10 mins with buffer.

For TAR2-5, clones supernatants from the round 2 selection werescreened.

48 clones were screened from each of the 3U, 5U and 7U libraries, usingthe following selection methods:

R1: 1 μg/ml human p55 TNF receptor immunotube, R2 1 μg/ml human p55 TNFreceptor immunotube, overnight wash.

R1: 10 μg/ml human p55 TNF receptor immunotube, R2 10 μg/ml human p55TNF receptor immunotube, overnight wash.

1.3.3 Receptor and Cell Assays

The ability of the dimers to neutralize in the receptor assay wasconducted as follows:

Receptor Binding

Anti-TNF dAbs were tested for the ability to inhibit the binding of TNFto recombinant TNF receptor 1 (p55). Briefly, Maxisorp plates wereincubated overnight with 30 mg/ml anti-human Fc mouse monoclonalantibody (Zymed, San Francisco, USA). The wells were washed withphosphate buffered saline (PBS) containing 0.05% Tween-20 and thenblocked with 1% BSA in PBS before being incubated with 100 ng/ml TNFreceptor 1 Fc fusion protein (R&D Systems, Minneapolis, USA). Anti-TNFdAb was mixed with TNF which was added to the washed wells at a finalconcentration of 10 ng/mi. TNF binding was detected with 0.2 mg/mlbiotinylated anti-TNF antibody (HyCult biotechnology, Uben, Netherlands)followed by 1 in 500 dilution of horse radish peroxidase labelledstreptavidin (Amersham Biosciences, UK) and then incubation with TMBsubstrate (KPL, Gaithersburg, USA). The reaction was stopped by theaddition of HCl and the absorbance was read at 450 nm. Anti-TNF dAbactivity lead to a decrease in TNF binding and therefore a decrease inabsorbance compared with the TNF only control.

L929 Cytotoxicity Assay

Anti-TNF dAbs were also tested for the ability to neutralise thecytotoxic activity of TNF on mouse L929 fibroblasts (Evans, T. (2000)Molecular Biotechnology 15, 243-248). Briefly, L929 cells plated inmicrotiter plates were incubated overnight with anti-TNF dAb, 100 pg/mlTNF and 1 mg/ml actinomycin D (Sigma, Poole, UK). Cell viability wasmeasured by reading absorbance at 490 nm following an incubation with[3-(4,5-dimethylthiazol-2-yl)-5-(3-carbboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(Promega, Madison, USA). Anti-TNF dAb activity lead to a decrease in TNFcytotoxicity and therefore an increase in absorbance compared with theTNF only control.

In the initial screen, supernatants prepared for Biacore analysis,described above, were also used in the receptor assay. Further analysisof selected dimers was also conducted in the receptor and cell assaysusing purified proteins.

HeLa IL-8 Assay

Anti-TNFR1 or anti-TNF alpha dAbs were tested for the ability toneutralize the induction of IL-8 secretion by TNF in HeLa cells (methodadapted from that of Akeson, L. et al (1996) Journal of BiologicalChemistry 271, 30517-30523, describing the induction of IL-8 by IL-1 inHUVEC; here we look at induction by human TNF alpha and we use HeLacells instead of the HUVEC cell line). Briefly, HeLa cells plated inmicrotitre plates were incubated overnight with dAb and 300 pg/ml TNF.Post incubation the supernatant was aspirated off the cells and IL-8concentration measured via a sandwich ELISA (R&D Systems). Anti-TNFR1dAb activity lead to a decrease in IL-8 secretion into the supernatantcompared with the TNF only control.

The L929 assay is used throughout the following experiments; however,the use of the HeLa IL-8 assay is preferred to measure anti-TNF receptor1 (p55) ligands; the presence of mouse p55 in the L929 assay posescertain limitations in its use.

1.4 Sequence Analysis

Dimers that proved to have interesting properties in the Biacore and thereceptor assay screens were sequenced. Sequences are detailed in thesequence listing.

1.5 Formatting 1.5.1 TAR1-5-19 Dimers

The TAR1-5 dimers that were shown to have good neutralisation propertieswere re-formatted and analysed in the cell and receptor assays. TheTAR1-5 guiding dAb was substituted with the affinity matured cloneTAR1-5-19. To achieve this TAR1-5 was cloned out of the individual dimerpair and substituted with TAR1-5-19 that had been amplified by PCR. Inaddition, TAR1-5-19 homodimers were also constructed in the 3U, 5U and7U vectors. The N terminal copy of the gene was amplified by PCR andcloned as described above and the C-terminal gene fragment was clonedusing existing Sal1 and Not1 restriction sites.

1.5.2 Mutagenesis

The amber stop codon present in dAb2, one of the C-terminal dAbs in theTAR1-5 dimer pairs was mutated to a glutamine by site-directedmutagenesis.

1.5.3 Fabs

The dimers containing TAR1-5 or TAR1-5-19 were re-formatted into Fabexpression vectors. dAbs were cloned into expression vectors containingeither the CK or CH genes using Sfi1 and Not1 restriction sites andverified by sequence analysis. The CK vector is derived from a pUC basedampicillin resistant vector and the CH vector is derived from a pACYCchloramphenicol resistant vector. For Fab expression the dAb-CH anddAb-CK constructs were co-transformed into HB2151 cells and grown in2×TY containing 0.1% glucose, 100 μg/ml ampicillin and 10 μg/mlchloramphenicol.

1.5.3 Hinge Dimerisation

Dimerisation of dAbs via cysteine bond formation was examined. A shortsequence of amino acids EPKSGDKTHTCPPCP a modified form of the humanIgGC1 hinge was engineered at the C terminal region on the dAb. An oligolinker encoding for this sequence was synthesised and annealed, asdescribed previously. The linker was cloned into the pEDA vectorcontaining TAR1-5-19 using Xho1 and Not1 restriction sites. Dimerisationoccurs in situ in the periplasm.

1.6 Expression and Purification 1.6.1 Expression

Supernatants were prepared in the 2 ml, 96-well plate format for theinitial screening as described previously. Following the initialscreening process selected dimers were analysed further. Dimerconstructs were expressed in TOP10F′ or HB2151 cells as supernatants.Briefly, an individual colony from a freshly streaked plate was grownovernight at 37° C. in 2×TY with 100 μg/ml ampicillin and 1% glucose. A1/100 dilution of this culture was inoculated into 2×TY with 100 μg/mlampicillin and 0.1% glucose and grown at 37° C. shaking until OD600 wasapproximately 0.9. The culture was then induced with 1 mM IPTG overnightat 30° C. The cells were removed by centrifugation and the supernatantpurified with protein A or L agarose.

Fab and cysteine hinge dimers were expressed as periplasmic proteins inHB2152 cells. A 1/100 dilution of an overnight culture was inoculatedinto 2×TY with 0.1% glucose and the appropriate antibiotics and grown at30° C. shaking until OD600 was approximately 0.9. The culture was theninduced with 1 mM IPTG for 3-4 hours at 25° C. The cells were harvestedby centrifugation and the pellet resuspended in periplasmic preparationbuffer (30 mM Tris-HCl pH8.0, 1 mM EDTA, 20% sucrose). Followingcentrifugation the supernatant was retained and the pellet resuspendedin 5 mM MgSO4. The supernatant was harvested again by centrifugation,pooled and purified.

1.6.2 Protein A/L Purification

Optimisation of the purification of dimer proteins from Protein Lagarose (Affitech, Norway) or Protein A agarose (Sigma, UK) wasexamined. Protein was eluted by batch or by column elution using aperistaltic pump. Three buffers were examined 0.1M Phosphate-citratebuffer pH2.6, 0.2M Glycine pH2.5 and 0.1M Glycine pH2.5. The optimalcondition was determined to be under peristaltic pump conditions using0.1M Glycine pH2.5 over 10 column volumes. Purification from protein Awas conducted peristaltic pump conditions using 0.1M Glycine pH2.5.

1.6.3 FPLC Purification

Further purification was carried out by FPLC analysis on the AKTAExplorer 100 system (Amersham Biosciences Ltd). TAR1-5 and TAR1-5-19dimers were fractionated by cation exchange chromatography (1 mlResource S—Amersham Biosciences Ltd) eluted with a 0-1M NaCl gradient in50 mM acetate buffer pH4. Hinge dimers were purified by ion exchange (1ml Resource Q Amersham Biosciences Ltd) eluted with a 0-1M NaCl gradientin 25 mMTris HCl pH 8.0. Fabs were purified by size exclusionchromatography using a superose 12 (Amersham Biosciences Ltd) column runat a flow rate of 0.5 ml/min in PBS with 0.05% tween. Followingpurification samples were concentrated using vivaspin 5K cut offconcentrators (Vivascience Ltd).

2.0 Results 2.1 TAR1-5 Dimers

6×96 clones were picked from the round 2 selection encompassing all thelibraries and selection conditions. Supernatant preps were made andassayed by antigen and Protein L ELISA, Biacore and in the receptorassays. In ELISAs, positive binding clones were identified from eachselection method and were distributed between 3U, 5U and 7U libraries.However, as the guiding dAb is always present, it was not possible todiscriminate between high and low affinity binders by this methodtherefore Biacore analysis was conducted.

Biacore analysis was conducted using the 2 ml supernatants. Biacoreanalysis revealed that the dimer Koff rates were vastly improvedcompared to monomeric TAR1-5. Monomer Koff rate was in the range of10⁻¹M compared with dimer Koff rates which were in the range of10⁻³-10⁻⁴M. Sixteen clones that appeared to have very slow off rateswere selected, these came from the 3U, 5U and 7U libraries and weresequenced. In addition, the supernatants were analysed for the abilityto neutralise human TNFα in the receptor assay.

6 lead clones (d1-d6 below) that neutralised in these assays and havebeen sequenced. The results shows that out of the 6 clones obtained,there are only 3 different second dAbs (dAb1, dAb2 and dAb3); howeverwhere the second dAb is found more than once they are linked withdifferent length linkers.

TAR1-5d1: 3U linker 2^(nd) dAb=dAb1−1 μg/ml Ag immunotube overnight washTAR1-5d2: 3U linker 2^(nd) dAb=dAb2−1 μg/ml Ag immunotube overnight washTAR1-5d3: 5U linker 2^(nd) dAb=dAb2−1 μg/ml Ag immunotube overnight washTAR1-5d4: 5U linker 2^(nd) dAb=dAb3−20 μg/ml Ag immunotube overnightwashTAR1-5d5: 5U linker 2^(nd) dAb=dAb1−20 μg/ml Ag immunotube overnightwashTAR1-5d6:7U linker 2^(nd) dAb=dAb1−R1:1 μg/ml Ag immunotube overnightwash, R2:beads

The six lead clones were examined further. Protein was produced from theperiplasm and supernatant, purified with protein L agarose and examinedin the cell and receptor assays. The levels of neutralisation werevariable (Table 1). The optimal conditions for protein preparation weredetermined. Protein produced from HB2151 cells as supernatants gave thehighest yield (approximately 10 mgs/L of culture). The supernatants wereincubated with protein L agarose for 2 hrs at room temperature orovernight at 4° C. The beads were washed with PBS/NaCl and packed ontoan FPLC column using a peristaltic pump. The beads were washed with 10column volumes of PBS/NaCl and eluted with 0.1M glycine pH2.5. Ingeneral, dimeric protein is eluted after the monomer.

TAR1-5d1-6 dimers were purified by FPLC. Three species were obtained, byFPLC purification and were identified by SDS PAGE. One speciescorresponds to monomer and the other two species corresponds to dimersof different sizes. The larger of the two species is possibly due to thepresence of C terminal tags. These proteins were examined in thereceptor assay. The data presented in the Table 1 represents the optimumresults obtained from the two dimeric species (FIG. 11).

The three second dAbs from the dimer pairs (i.e., dAb1, dAb2 and dAb3)were cloned as monomers and examined by ELISA and in the cell andreceptor assay. All three dAbs bind specifically to TNF by antigen ELISAand do not cross react with plastic or BSA. As monomers, none of thedAbs neutralise in the cell or receptor assays.

2.1.2 TAR1-5-19 Dimers

TAR1-5-19 was substituted for TAR1-5 in the six lead clones. Analysis ofall TAR1-5-19 dimers in the cell and receptor assays was conducted usingtotal protein (protein L purified only) unless otherwise stated (Table2). TAR1-5-19d4 and TAR1-5-19d3 have the best ND₅₀ (˜5 nM) in the cellassay, this is consistent with the receptor assay results and is animprovement over TAR1-5-19 monomer (ND₅₀˜30 nM). Although purifiedTAR1-5 dimers give variable results in the receptor and cell assays,TAR1-5-19 dimers were more consistent. Variability was shown when usingdifferent elution buffers during the protein purification. Elution using0.1 M Phosphate-citrate buffer pH2.6 or 0.2 M Glycine pH2.5, althoughremoving all protein from the protein L agarose in most cases renderedit less functional.

TAR1-5-19d4 was expressed in the fermenter and purified on cationexchange FPLC to yield a completely pure dimer. As with TAR1-5d4 threespecies were obtained, by FPLC purification corresponding to monomer andtwo dimer species. This dimer was amino acid sequenced. TAR1-5-19monomer and TAR1-5-19d4 were then examined in the receptor assay and theresulting IC50 for monomer was 30 nM and for dimer was 8 nM. The resultsof the receptor assay comparing TAR1-5-19 monomer, TAR1-5-19d4 andTAR1-5d4 is shown in FIG. 10.

TAR1-5-19 homodimers were made in the 3U, 5U and 7U vectors, expressedand purified on Protein L. The proteins were examined in the cell andreceptor assays and the resulting IC₅₀s (for receptor assay) and NDsos(for cell assay) were determined (Table 3, FIG. 12).

2.2 Fabs

TAR1-5 and TAR1-5-19 dimers were also cloned into Fab format, expressedand purified on protein L agarose. Fabs were assessed in the receptorassays (Table 4). The results showed that for both TAR1-5-19 and TAR1-5dimers the neutralisation levels were similar to the original Gly₄Serlinker dimers from which they were derived. A TAR1-5-19 Fab whereTAR1-5-19 was displayed on both CH and CK was expressed, protein Lpurified and assessed in the receptor assay. The resulting IC50 wasapproximately 1 nM.

2.3 TAR1-27 Dimers

3×96 clones were picked from the round 2 selection encompassing all thelibraries and selection conditions. 2 ml supernatant preps were made foranalysis in ELISA and bioassays. Antigen ELISA gave 71 positive clones.The receptor assay of crude supernatants yielded 42 clones withinhibitory properties (TNF binding 0-60%). In the majority of casesinhibitory properties correlated with a strong ELISA signal. 42 cloneswere sequenced, 39 of these have unique second dAb sequences. The 12dimers that gave the best inhibitory properties were analysed further.

The 12 neutralising clones were expressed as 200 ml supernatant prepsand purified on protein L. These were assessed by protein L and antigenELISA, Biacore and in the receptor assay. Strong positive ELISA signalswere obtained in all cases. Biacore analysis revealed all clones to havefast on and off rates. The off rates were improved compared to monomericTAR1-27, however the off rate of TAR1-27 dimers was faster (Koff isapproximately in the range of 10⁻¹ and 10⁻²M) than the TAR1-5 dimersexamined previously (Koff is approximately in the range of 10⁻³-10⁻⁴M).The stability of the purified dimers was questioned and therefore inorder to improve stability, the addition on 5% glycerol, 0.5% TritonX100 or 0.5% NP40 (Sigma) was included in the purification of 2 TAR1-27dimers (d2 and d16). Addition of NP40 or Triton X100™ improved the yieldof purified product approximately 2 fold. Both dimers were assessed inthe receptor assay. TAR1-27d2 gave IC50 of ˜30 nM under all purificationconditions. TAR1-27d16 showed no neutralisation effect when purifiedwithout the use of stabilising agents but gave an IC50 of ˜50 nM whenpurified under stabilising conditions. No further analysis wasconducted.

2.4 TAR2-5 Dimers

3×96 clones were picked from the second round selections encompassingall the libraries and selection conditions. 2 ml supernatant preps weremade for analysis. Protein A and antigen ELISAs were conducted for eachplate. 30 interesting clones were identified as having good off-rates byBiacore (Koff ranges between 10⁻²-10⁻³M). The clones were sequenced and13 unique dimers were identified by sequence analysis.

TABLE 1 TAR1-5 dimers Receptor/ Cell Protein Elution Cell Dimer typePurification Fraction conditions assay TAR1-5d1 HB2151 Protein L + small0.1M glycine RA~30 nM FPLC dimeric pH 2.5 species TAR1-5d2 HB2151Protein L + small 0.1M glycine RA~50 nM FPLC dimeric pH 2.5 speciesTAR1-5d3 HB2151 Protein L + large 0.1M glycine RA~300 nM FPLC dimeric pH2.5 species TAR1-5d4 HB2151 Protein L + small 0.1M glycine RA~3 nM FPLCdimeric pH 2.5 species TAR1-5d5 HB2151 Protein L + large 0.1M glycineRA~200 nM FPLC dimeric pH 2.5 species TAR1-5d6 HB2151 Protein L + Large0.1M glycine RA~100 nM FPLC dimeric pH 2.5 species *note dimer 2 anddimer 3 have the same second dAb (called dAb2), however have differentlinker lengths (d2 = (Gly₄Ser)₃, d3 = (Gly₄Ser)₃). dAb1 is the partnerdAb to dimers 1, 5 and 6. dAb3 is the partner dAb to dimer4. None of thepartner dAbs neutralise alone. FPLC purification is by cation exchangeunless otherwise stated. The optimal dimeric species for each dimerobtained by FPLC was determined in these assays.

TABLE 2 TAR1-5-19 dimers Receptor/ Cell Protein Elution Cell Dimer typePurification Fraction conditions assay TAR1-5-19 d1 TOP10F′ Protein LTotal protein 0.1M glycine pH RA~15 nM 2.0 TAR1-5-19 d2 TOP10F′ ProteinL Total protein 0.1M glycine pH RA~2 nM (no stop codon) 2.0 + 0.05% NP40TAR1-5-19d3 TOP10F′ Protein L Total protein 0.1M glycine pH RA~8 nM (nostop codon) 2.5 + 0.05% NP40 TAR1-5-19d4 TOP10F′ Protein L + FPLCpurified 0.1M glycine RA~2-5 nM FPLC fraction pH 2.0 CA~12 nMTAR1-5-19d5 TOP10F′ Protein L Total protein 0.1M glycine RA~8 nM pH2.0 + NP40 CA~10 nM TAR1-5-19d6 TOP10F′ Protein L Total protein 0.1Mglycine pH RA~10 nM 2.0

TABLE 3 TAR1-5-19 homodimers Elution Receptor/ Dimer Cell typePurification Protein Fraction conditions Cell assay TAR1-5-19 3U HB2151Protein L Total protein 0.1M glycine pH 2.5 RA~20 nM homodimer CA~30 nMTAR1-5-19 5U HB2151 Protein L Total protein 0.1M glycine pH 2.5 RA~2 nMhomodimer CA~3 nM TAR1-5-19 7U HB2151 Protein L Total protein 0.1Mglycine pH 2.5 RA~10 nM homodimer CA~15 nM TAR1-5-19 cys HB2151 ProteinL + FPLC FPLC purified 0.1M glycine pH 2.5 RA~2 nM hinge dimer fractionTAR1-5-19CH/ HB2151 Protein Total protein 0.1M glycine pH 2.5 RA~1 nMTAR1-5-19 CK

TABLE 4 TAR1-5/TAR1-5-19 Fabs Elution Receptor/ Dimer Cell typePurification Protein Fraction conditions Cell assay TAR1-5CH/ HB2151Protein L Total protein 0.1M citrate pH 2.6 RA~90 nM dAb1 CK TAR1-5CH/HB2151 Protein L Total protein 0.1M glycine pH 2.5 RA~30 nM dAb2 CKCA~60 nM dAb3CH/ HB2151 Protein L Total protein 0.1M citrate pH 2.6RA~100 nM TAR1-5CK TAR1-5-19CH/ HB2151 Protein L Total protein 0.1Mglycine pH 2.0 RA~6 nM dAb1 CK dAb1 CH/ HB2151 Protein L 0.1M glycineMyc/flag RA~6 nM TAR1-5-19CK pH 2.0 TAR1-5-19CH/ HB2151 Protein L Totalprotein 0.1M glycine pH 2.0 RA~8 nM dAb2 CK CA~12 nM TAR1-5-19CH/ HB2151Protein L Total protein 0.1M glycine pH 2.0 RA~3 nM dAb3CK

Example 7 dAb Dimerisation by Terminal Cysteine Linkage Summary

For dAb dimerisation, a free cysteine has been engineered at theC-terminus of the protein. When expressed the protein forms a dimerwhich can be purified by a two step purification method.

PCR Construction of TAR1-5-19CYS Dimer

See example 8 describing the dAb trimer. The trimer protocol gives riseto a mixture of monomer, dimer and trimer.

Expression and Purification of TAR1-5-19CYS Dimer

The dimer was purified from the supernatant of the culture by capture onProtein L agarose as outlined in the example 8.

Separation of TAR1-5-19CYS monomer from the TAR1-5-19CYS dimer

Prior to cation exchange separation, the mixed monomer/dimer sample wasbuffer exchanged into 50 mM sodium acetate buffer pH 4.0 using a PD-10column (Amersham Pharmacia), following the manufacturer's guidelines.The sample was then applied to a 1 mL Resource S cation exchange column(Amersham Pharmacia), which had been pre-equilibrated with 50 mM sodiumacetate pH 4.0. The monomer and dimer were separated using the followingsalt gradient in 50 mM sodium acetate pH 4.0:

150 to 200 mM sodium chloride over 15 column volumes200 to 450 mM sodium chloride over 10 column volumes450 to 1000 mM sodium chloride over 15 column volumes

Fractions containing dimer only were identified using SDS-PAGE and thenpooled and the pH increased to 8 by the addition of ⅕ volume of 1M TrispH 8.0.

In vitro functional binding assay: TNF receptor assay and cell assay

The affinity of the dimer for human TNFα was determined using the TNFreceptor and cell assay. IC50 in the receptor assay was approximately0.3-0.8 nM; ND50 in the cell assay was approximately 3-8 nM.

Other possible TAR1-5-19CYS dimer formats

PEG Dimers and Custom Synthetic Maleimide Dimers

Nektar (Shearwater) offer a range of bi-maleimide PEGs[mPEG2-(MAL)2 ormPEG-(MAL)2] which would allow the monomer to be formatted as a dimer,with a small linker separating the dAbs and both being linked to a PEGranging in size from 5 to 40 Kda. It has been shown that the 5 KdamPEG-(MAL)2 (i.e., [TAR1-5-19]-Cys-maleimide-PEG×2, wherein themaleimides are linked together in the dimer) has an affinity in the TNFreceptor assay of ˜1-3 nM. Also the dimer can also be produced usingTMEA (Tris[2-maleimidoethyl]amine) (Pierce Biotechnology) or otherbi-functional linkers.

It is also possible to produce the disulphide dimer using a chemicalcoupling procedure using 2,2′-dithiodipyridine (Sigma Aldrich) and thereduced monomer.

Addition of a Polypeptide Linker or Hinge to the C-Terminus of the dAb.

A small linker, either (Gly₄Ser)n where n=1 to 10, e.g., 1, 2, 3, 4, 5,6 or 7, an immunoglobulin (e.g., IgG hinge region or random peptidesequence (e.g., selected from a library of random peptide sequences) canbe engineered between the dAb and the terminal cysteine residue. Thiscan then be used to make dimers as outlined above.

Example 8 dAb Trimerisation Summary

For dAb trimerisation, a free cysteine is required at the C-terminus ofthe protein. The cysteine residue, once reduced to give the free thiol,can then be used to specifically couple the protein to a trimericmaleimide molecule, for example TMEA (Tris[2-maleimidoethyl]amine).

PCR Construction of TAR1-5-19CYS

The following oligonucleotides were used to specifically PCR TAR1-5-19with a SalI and BamHI sites for cloning and also to introduce aC-terminal cysteine residue:

           SalI           ~~~~~~~~   1Trp Ser Ala Ser Thr Asp* Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValTGG AGC GCG TCG ACG GAC ATC CAG ATG ACC CAG TCT CCA TCC TCT CTG TCT GCA TCT GTA ACC TCGCGC AGC TGC CTG TAG GTC TAC TGG GTC AGA GGT  AGG AGA GAC AGA CGT AGA CAT 61 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr Leu His TrpGGA GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAT AGT TAT TTA CAT TGG CCT CTGGCA CAG TGG TAG TGA ACG GCC CGT TCA GTC TCG  TAA CTA TCA ATA AAT GTA ACC121 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Glu Leu GlnTAC CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC TAT AGT GCA TCC GAG TTG CAA ATG GTCGTC TTT GGT CCC TTT CGG GGA TTC GAG GAC TAG  ATA TCA CGT AGG CTC AAC GTT181 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr IleAGT GGG GTC CCA TCA CGT TTC AGT GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC TCA CCCCAG GGT AGT GCA AAG TCA CCG TCA CCT AGA CCC  TGT CTA AAG TGA GAG TGG TAG241 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg ProAGC AGT CTG CAA CCT GAA GAT TTT GCT ACG TAC TAC TGT CAA CAG GTT GTG TGG CGT CCT TCG TCAGAC GTT GGA CTT CTA AAA CGA TGC ATG ATG ACA  GTT GTC CAA CAC ACC GCA GGA           BamHI            ~~~~~~~~ 301Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Cys *** *** Gly Ser GlyTTT ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG TGC TAA TAA GGA TCC GGC AAA TGC AAGCCG GTT CCC TGG TTC CAC CTT TAG TTT GCC ACG  ATT ATT CCT AGG CCG(* start of TAR1-5-19CYS sequence) Forward primer 5′-TGGAGCGCGTCGACGGACATCCAGATGACCCAGTCTCCA-3′ Reverse primer5′-TTAGCAGCCGGATCCTTATTAGCACCGTTTGATTTCCAC-3′

The PCR reaction (50 μL volume) was set up as follows: 200 μM dNTPs, 0.4μM of each primer, 5 μL of 10×PfuTurbo buffer (Stratagene), 100 ng oftemplate plasmid (encoding TAR1-5-19), 1 μL of PfuTurbo enzyme(Stratagene) and the volume adjusted to 50 μL using sterile water. Thefollowing PCR conditions were used: initial denaturing step 94° C. for 2mins, then 25 cycles of 94° C. for 30 secs, 64° C. for 30 sec and 72° C.for 30 sec. A final extension step was also included of 72° C. for 5mins. The PCR product was purified and digested with SalI and BamHI andligated into the vector which had also been cut with the samerestriction enzymes. Correct clones were verified by DNA sequencing.

Expression and Purification of TAR1-5-19CYS

TAR1-5-19CYS vector was transformed into BL21 (DE3) pLysS chemicallycompetent cells (Novagen) following the manufacturer's protocol. Cellscarrying the dAb plasmid were selected for using 100 μg/mL carbenicillinand 37 μg/mL chloramphenicol. Cultures were set up in 2 L baffled flaskscontaining 500 mL of terrific broth (Sigma-Aldrich), 100 μg/mLcarbenicillin and 37 μg/mL chloramphenicol. The cultures were grown at30° C. at 200 rpm to an O.D.600 of 1-1.5 and then induced with 1 mM IPTG(isopropyl-beta-D-thiogalactopyranoside, from Melford Laboratories). Theexpression of the dAb was allowed to continue for 12-16 hrs at 30° C. Itwas found that most of the dAb was present in the culture media.Therefore, the cells were separated from the media by centrifugation(8,000×g for 30 mins), and the supernatant used to purify the dAb. Perlitre of supernatant, 30 mL of Protein L agarose (Affitech) was addedand the dAb allowed to batch bind with stirring for 2 hours. The resinwas then allowed to settle under gravity for a further hour before thesupernatant was siphoned off. The agarose was then packed into a XK 50column (Amersham Pharmacia) and was washed with 10 column volumes ofPBS. The bound dAb was eluted with 100 mM glycine pH 2.0 and proteincontaining fractions were then neutralized by the addition of ⅕ volumeof 1 M Tris pH 8.0. Per litre of culture supernatant 20 mg of pureprotein was isolated, which contained a 50:50 ratio of monomer to dimer.

Trimerisation of TAR1-5-19CYS

2.5 ml of 100 μM TAR1-5-19CYS was reduce with 5 mM dithiothreitol andleft at room temperature for 20 minutes. The sample was then bufferexchanged using a PD-10 column (Amersham Pharmacia). The column had beenpre-equilibrated with 5 mM EDTA, 50 mM sodium phosphate pH 6.5, and thesample applied and eluted following the manufactures guidelines. Thesample was placed on ice until required. TMEA(Tris[2-maleimidoethyl]amine) was purchased from Pierce Biotechnology. A20 mM stock solution of TMEA was made in 100% DMSO (dimethylsulphoxide). It was found that a concentration of TMEA greater than 3:1(molar ratio of dAb:TMEA) caused the rapid precipitation andcross-linking of the protein. Also the rate of precipitation andcross-linking was greater as the pH increased. Therefore using 100 μMreduced TAR1-5-19CYS, 25 μM TMEA was added to trimerise the protein andthe reaction allowed to proceed at room temperature for two hours. Itwas found that the addition of additives such as glycerol or ethyleneglycol to 20% (v/v), significantly reduced the precipitation of thetrimer as the coupling reaction proceeded. After coupling, SDS-PAGEanalysis showed the presence of monomer, dimer and trimer in solution.

Purification of the Trimeric TAR1-5-19CYS

40 μL of 40% glacial acetic acid was added per mL of theTMEA-TAR1-5-19cys reaction to reduce the pH to ˜4. The sample was thenapplied to a 1 mL Resource S cation exchange column (AmershamPharmacia), which had been pre-equilibrated with 50 mM sodium acetate pH4.0. The dimer and trimer were partially separated using a salt gradientof 340 to 450 mM Sodium chloride, 50 mM sodium acetate pH 4.0 over 30column volumes. Fractions containing trimer only were identified usingSDS-PAGE and then pooled and the pH increased to 8 by the addition of ⅕volume of 1M Tris pH 8.0. To prevent precipitation of the trimer duringconcentration steps (using 5K cut off Viva spin concentrators;Vivascience), 10% glycerol was added to the sample.

In Vitro Functional Binding Assay: TNF Receptor Assay and Cell Assay

The affinity of the trimer for human TNFα was determined using the TNFreceptor and cell assay. IC50 in the receptor assay was 0.3 nM; ND50 inthe cell assay was in the range of 3 to 10 nM (e.g., 3 nM).

Other Possible TAR1-5-19CYS Trimer Formats

TAR1-5-19CYS may also be formatted into a trimer using the followingreagents:

PEG Trimers and Custom Synthetic Maleimide Trimers

Nektar (Shearwater) offer a range of multi arm PEGs, which can bechemically modified at the terminal end of the PEG. Therefore using aPEG trimer with a maleimide functional group at the end of each armwould allow the trimerisation of the dAb in a manner similar to thatoutlined above using TMEA. The PEG may also have the advantage inincreasing the solubility of the trimer thus preventing the problem ofaggregation. Thus, one could produce a dAb trimer in which each dAb hasa C-terminal cysteine that is linked to a maleimide functional group,the maleimide functional groups being linked to a PEG trimer.

Addition of a Polypeptide Linker or Hinge to the C-Terminus of the dAb

A small linker, either (Gly₄Ser)_(n) where n=1 to 10, e.g., 1, 2, 3, 4,5, 6 or 7, an immunoglobulin (e.g., IgG hinge region or random peptidesequence (e.g., selected from a library of random peptide sequences)could be engineered between the dAb and the terminal cysteine residue.When used to make multimers (e.g., dimers or trimers), this again wouldintroduce a greater degree of flexibility and distance between theindividual monomers, which may improve the binding characteristics tothe target, e.g., a multisubunit target such as human TNFα.

Example 9 Selection of a Collection of Single Domain Antibodies (dAbs)Directed Against Human Serum Albumin (HSA) and Mouse Serum Albumin (MSA)

This example explains a method for making a single domain antibody (dAb)directed against serum albumin. Selection of dAbs against both mouseserum albumin (MSA) and human serum albumin (HSA) is described. Threehuman phage display antibody libraries were used in this experiment,each based on a single human framework for V_(H) (see FIG. 13: sequenceof dummy V_(H) based on V3-23/DP47 and JH4b) or V_(κ) (see FIG. 15:sequence of dummy V_(κ) based on o12/o2/DPK9 and Jk1) with side chaindiversity encoded by NNK codons incorporated in complementaritydetermining regions (CDR1, CDR2 and CDR3).

Library 1 (V_(H)):

Diversity at positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55,H56, H58, H95, H97, H98.

Library size: 6.2×10⁹

Library 2 (V_(H)):

Diversity at positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55,H56, H58, H95, H97, H98, H99, H100, H100a, H100b.

Library size: 4.3×10⁹

Library 3 (V_(κ)):

Diversity at positions: L30, L31, L32, L34, L50, L53, L91, L92, L93,L94, L96

Library size: 2×10⁹

The V_(H) and V_(κ) libraries have been preselected for binding togeneric ligands protein A and protein L respectively so that themajority of clones in the unselected libraries are functional. The sizesof the libraries shown above correspond to the sizes after preselection.

Two rounds of selection were performed on serum albumin using each ofthe libraries separately. For each selection, antigen was coated onimmunotube (nunc) in 4 ml of PBS at a concentration of 100 μg/ml. In thefirst round of selection, each of the three libraries was pannedseparately against HSA (Sigma) and MSA (Sigma). In the second round ofselection, phage from each of the six first round selections was pannedagainst (i) the same antigen again (e.g. 1^(st) round MSA, 2^(nd) roundMSA) and (ii) against the reciprocal antigen (e.g. 1^(st) round MSA,2^(nd) round HSA) resulting in a total of twelve 2^(nd) roundselections. In each case, after the second round of selection 48 cloneswere tested for binding to HSA and MSA. Soluble dAb fragments wereproduced as described for scFv fragments by Harrison et al, MethodsEnzymol. 1996; 267:83-109 and standard ELISA protocol was followed(Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133) except that 2%tween PBS was used as a blocking buffer and bound dAbs were detectedwith either protein L-HRP (Sigma) (for the V_(κS)) and protein A-HRP(Amersham Pharmacia Biotech) (for the V_(HS)).

dAbs that gave a signal above background indicating binding to MSA, HSAor both were tested in ELISA insoluble form for binding to plastic alonebut all were specific for serum albumin. Clones were then sequenced (seeTable 5) revealing that 21 unique dAb sequences had been identified. Theminimum similarity (at the amino acid level) between the V_(κ) dAbclones selected was 86.25% ((69/80)×100; the result when all thediversified residues are different, e.g. clones 24 and 34). The minimumsimilarity between the V_(H) dAb clones selected was 94%((127/136)×100).

Next, the serum albumin binding dAbs were tested for their ability tocapture biotinylated antigen from solution. ELISA protocol (as above)was followed except that ELISA plate was coated with 1 μg/ml protein L(for the V_(κ) clones) and 1 μg/ml protein A (for the V_(H) clones).Soluble dAb was captured from solution as in the protocol and detectionwas with biotinylated MSA or HSA and streptavidin HRP. The biotinylatedMSA and HSA had been prepared according to the manufacturer'sinstructions, with the aim of achieving an average of 2 biotins perserum albumin molecule. Twenty four clones were identified that capturedbiotinylated MSA from solution in the ELISA, Table 5. Two of these(clones 2 and 38 below) also captured biotinylated HSA. Next, the dAbswere tested for their ability to bind MSA coated on a CM5 Biacore chip.Eight clones were found that bound MSA on the Biacore.

TABLE 5 dAb (all Binds capture MSA Captures biotinylated H inbiotinylated MSA) or κ CDR1 CDR2 CDR3 Biacore HSA Vκ library 3 κ XXXLXXASXLQS QQXXXXPXT template (dummy)  2, 4, 7, 41, κ SSYLN RASPLQSQQTYSVPPT ✓ all 4 bind 38, 54 κ SSYLN RASPLQS QQTYRIPPT ✓ both bind46, 47, 52, κ FKSLK NASYLQS QQVVYWPVT 56 13, 15 κ YYHLK KASTLQSQQVRKVPRT 30, 35 κ RRYLK QASVLQS QQGLYPPIT 19, κ YNWLK RASSLQS QQNVVIPRT22, κ LWHLR HASLLQS QQSAVYPKT 23, κ FRYLA HASHLQS QQRLLYPKT 24, κ FYHLAPASKLQS QQRARWPRT 31, κ IWHLN RASRLQS QQVARVPRT 33, κ YRYLR KASSLQSQQYVGYPRT 34, κ LKYLK NASHLQS QQTTYYPIT 53, κ LRYLR KASWLQS QQVLYYPQT11, κ LRSLK AASRLQS QQVVYWPAT ✓ 12, κ FRHLK AASRLQS QQVALYPKT ✓ 17, κRKYLR TASSLQS QQNLFWPRT ✓ 18, κ RRYLN AASSLQS QQMLFYPKT ✓ 16, 21 κ IKHLKGASRLQS QQGARWPQT ✓ 25, 26 κ YYHLK KASTLQS QQVRKVPRT ✓ 27, κ YKHLKNASHLQS QQVGRYPKT ✓ 55, κ FKSLK NASYLQS QQVVYWPVT ✓ V_(H) library 1 HXXYXXX XIXXXGXXTXYADSVK XXXX(XXXX)FDY (and 2) G template (dummy)  8, 10H WVYQMD SISAFGAKTLYADSVK LSGKFDY G 36, H WSYQMT SISSFGSSTLYADSVKGRDHNYSLFDY G

In all cases the frameworks were identical to the frameworks in thecorresponding dummy sequence, with diversity in the CDRs as indicated inTable 5.

Of the eight clones that bound MSA on the Biacore, two clones that arehighly expressed in E. coli (clones MSA16 and MSA26) were chosen forfurther study (see Example 10). Full nucleotide and amino acid sequencesfor MSA16 and 26 are given in FIG. 16.

Example 10 Determination of Affinity and Serum Half-Life in Mouse of MSABinding dAbs MSA16 and MSA26

As described in US20060251644, one common method for determining bindingaffinity is by assessing the association and dissociation rate constantsusing a Biacore™ surface plasmon resonance system (Biacore, Inc.). Abiosensor chip is activated for covalent coupling of the targetaccording to the manufacturer's (Biacore) instructions. The target isthen diluted and injected over the chip to obtain a signal in responseunits (RU) of immobilized material. Since the signal in RU isproportional to the mass of immobilized material, this represents arange of immobilized target densities on the matrix. Dissociation dataare fit to a one-site model to obtain k_(off)+/−s.d. (standard deviationof measurements). Pseudo-first order rate constant (Kd's) are calculatedfor each association curve, and plotted as a function of proteinconcentration to obtain k_(off)+/−s.e. (standard error of fit).Equilibrium dissociation constants for binding, Kd's, are calculatedfrom SPR measurements as k_(off)/k_(on).

dAbs MSA16 and MSA26 were expressed in the periplasm of E. coli andpurified using batch absorption to protein L-agarose affinity resin(Affitech, Norway) followed by elution with glycine at pH 2.2. Thepurified dAbs were then analysed by inhibition Biacore to determine Kd.Briefly, purified MSA16 and MSA26 were tested to determine theconcentration of dAb required to achieve 200 RUs of response on aBiacore CM5 chip coated with a high density of MSA. Once the requiredconcentrations of dAb had been determined, MSA antigen at a range ofconcentrations around the expected Kd was premixed with the dAb andincubated overnight. Binding to the MSA coated Biacore chip of dAb ineach of the premixes was then measured at a high flow-rate of 30μl/minute. The affinities are determined using surface plasmon resonance(SPR) and the Biacore (Karlsson et al., 1991). The Biacore system(Uppsala, Sweden) is a preferred method for determining bindingaffinity. The Biacore system uses surface plasmon resonance (SPR,Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998,Methods in Enzymology 295: 268) to monitor biomolecular interactions inreal time. Biacore analysis conveniently generates association rateconstants, dissociation rate constants, equilibrium dissociationconstants, and affinity constants. The resulting curves were used tocreate Klotz plots, (Klotz, I. M. (1982) Science 217:1247-1249 andKlotz, I. M. (1983) J. Trends in Pharmacol. Sci. 4:253-255) which gavean estimated Kd of 200 nM for MSA16 and 70 nM for MSA 26 (FIGS. 17 A &B).

Next, clones MSA16 and MSA26 were cloned into an expression vector withthe HA tag (nucleic acid sequence: TATCCTTATGATGTTCCTGATTATGCA and aminoacid sequence: YPYDVPDYA) and 2-10 mg quantities were expressed in E.coli and purified from the supernatant with protein L-agarose affinityresin (Affitech, Norway) and eluted with glycine at pH2.2. Serum halflife of the dAbs was determined in mouse. MSA26 and MSA16 were dosed assingle i.v. injections at approx 1.5 mg/kg into CD1 mice. Analysis ofserum levels was by goat anti-HA (Abcam, UK) capture and protein L-HRP(invitrogen) detection ELISA which was blocked with 4% Marvel. Washingwas with 0.05% tween PBS. Standard curves of known concentrations of dAbwere set up in the presence of 1× mouse serum to ensure comparabilitywith the test samples. Modelling with a 2 compartment model showedMSA-26 had a t1/2α of 0.16 hr, a t1/2β of 14.5 hr and an area under thecurve (AUC) of 465 hr·mg/ml (data not shown) and MSA-16 had a t1/2a of0.98 hr, a t1/2β of 36.5 hr and an AUC of 913 hr·mg/ml (FIG. 18). Bothanti-MSA clones had considerably lengthened half life compared with HEL4(an anti-hen egg white lysozyme dAb) which had a t1/2α of 0.06 hr, and at1/2β of 0.34 hr.

Example 11. Creation of V_(H)-V_(H) and V_(κ)-V_(κ) Dual Specific FabLike Fragments

This example describes a method for making V_(H)-V_(H) and V_(κ)-V_(κ)dual specifics as Fab like fragments. Before constructing each of theFab like fragments described, dAbs that bind to targets of choice werefirst selected from dAb libraries similar to those described in example9. A V_(H) dAb, HEL4, that binds to hen egg lysozyme (Sigma) wasisolated and a second V_(H) dAb (TAR2h-5) that binds to TNFα receptor (Rand D systems) was also isolated. The sequences of these are given inthe sequence listing. A V_(κ) dAb that binds TNFα (TAR1-5-19) wasisolated by selection and affinity maturation and the sequence is alsoset forth in the sequence listing. A second V_(κ) dAb (MSA 26) describedin example 9 whose sequence is in FIG. 17B was also used in theseexperiments.

DNA from expression vectors containing the four dAbs described above wasdigested with enzymes SalI and NotI to excise the DNA coding for thedAb. A band of the expected size (300-400 bp) was purified by runningthe digest on an agarose gel and excising the band, followed by gelpurification using the Qiagen gel purification kit (Qiagen, UK). The DNAcoding for the dAbs was then inserted into either the C_(H) or C_(κ)vectors (FIGS. 8 and 9) as indicated in Table 6.

TABLE 6 dAb V_(H) or Inserted into tag (C Antibiotic dAb Target antigendAb Vκ vector terminal) resisitance HEL4 Hen egg lysozyme V_(H) C_(H)myc Chloramphenicol TAR2-5 TNF receptor V_(H) Cκ flag AmpicillinTAR1-5-19 TNF α Vκ C_(H) myc Chloramphenicol MSA 26 Mouse serum albuminVκ Cκ flag Ampicillin

The V_(H) C_(H) and V_(H) C_(κ) constructs were cotransformed intoHB2151 cells. Separately, the V_(κ) C_(H) and V_(κ) C_(κ) constructswere cotransformed into HB2151 cells. Cultures of each of thecotransformed cell lines were grown overnight (in 2×Ty containing 5%glucose, 10 μg/ml chloramphenicol and 100 μg/ml ampicillin to maintainantibiotic selection for both C_(H) and C_(κ) plasmids). The overnightcultures were used to inoculate fresh media (2×Ty, 10 μg/mlchloramphenicol and 100 μg/ml ampicillin) and grown to OD 0.7-0.9 beforeinduction by the addition of IPTG to express their C_(H) and C_(κ)constructs. Expressed Fab like fragment was then purified from theperiplasm by protein A purification (for the cotransformed V_(H) C_(H)and V_(H) C_(κ) and MSA affinity resin purification (for thecotransformed V_(κ) C_(H) and V_(κ) C_(κ)).

V_(H)-V_(H) Dual Specific

Expression of the V_(H) C_(H) and V_(H) C_(κ) dual specific was testedby running the protein on a gel. The gel was blotted and a band theexpected size for the Fab fragment could be detected on the Western blotvia both the myc tag and the flag tag, indicating that both the V_(H)C_(H) and V_(H) C_(κ) parts of the Fab like fragment were present. Next,in order to determine whether the two halves of the dual specific werepresent in the same Fab-like fragment, an ELISA plate was coatedovernight at 4° C. with 100 μl per well of hen egg lysozyme (HEL) at 3mg/ml in sodium bicarbonate buffer. The plate was then blocked (asdescribed in example 1) with 2% tween PBS followed by incubation withthe V_(H) C_(H)/V_(H) C_(κ) dual specific Fab like fragment. Detectionof binding of the dual specific to the HEL was via the non cognate chainusing 9e10 (a monoclonal antibody that binds the myc tag, Roche) andanti mouse IgG-HRP (Amersham Pharmacia Biotech). The signal for theV_(H) CH/V_(H) C_(κ) dual specific Fab like fragment was 0.154 comparedto a background signal of 0.069 for the V_(H) C_(κ) chain expressedalone. This demonstrates that the Fab like fragment has bindingspecificity for target antigen.

V_(κ)-V_(κ) Dual Specific

After purifying the cotransformed V_(κ) C_(H) and V_(κ) C_(κ) dualspecific Fab like fragment on an MSA affinity resin, the resultingprotein was used to probe an ELISA plate coated with 1 μg/ml TNFα and anELISA plate coated with 10 μg/ml MSA. As predicted, there was signalabove background when detected with protein L-HRP on both ELISA plates(data not shown). This indicated that the fraction of protein able tobind to MSA (and therefore purified on the MSA affinity column) was alsoable to bind TNFα in a subsequent ELISA, confirming the dual specificityof the antibody fragment. This fraction of protein was then used for twosubsequent experiments. Firstly, an ELISA plate coated with 1 μg/ml TNFαwas probed with dual specific V_(κ) C_(H) and V_(κ) C_(κ) Fab likefragment and also with a control TNFα binding dAb at a concentrationcalculated to give a similar signal on the ELISA. Both the dual specificand control dAb were used to probe the ELISA plate in the presence andin the absence of 2 mg/ml MSA. The signal in the dual specific well wasreduced by more than 50% but the signal in the dAb well was not reducedat all (see FIG. 19a ). The same protein was also put into the receptorassay with and without MSA and competition by MSA was also shown (seeFIG. 19c ). This demonstrates that binding of MSA to the dual specificis competitive with binding to TNFα.

Example 12. Creation of a V_(κ)-V_(κ) Dual Specific Cys Bonded DualSpecific with Specificity for Mouse Serum Albumin and TNFα

This example describes a method for making a dual specific antibodyfragment specific for both mouse serum albumin and TNFα by chemicalcoupling via a disulphide bond. Both MSA16 (from example 1) andTAR1-5-19 dAbs were recloned into a pET based vector with a C terminalcysteine and no tags. The two dAbs were expressed at 4-10 mg levels andpurified from the supernatant using protein L-agarose affinity resin(Affitiech, Norway). The cysteine tagged dAbs were then reduced withdithiothreitol. The TAR1-5-19 dAb was then coupled with dithiodipyridineto block reformation of disulphide bonds resulting in the formation ofPEP 1-5-19 homodimers. The two different dAbs were then mixed at pH 6.5to promote disulphide bond formation and the generation of TAR1-5-19,MSA16 cys bonded heterodimers. This method for producing conjugates oftwo unlike proteins was originally described by King et al. (King T P,Li Y Kochoumian L Biochemistry. 1978 vol 17:1499-506 Preparation ofprotein conjugates via intermolecular disulfide bond formation.)Heterodimers were separated from monomeric species by cation exchange.Separation was confirmed by the presence of a band of the expected sizeon a SDS gel. The resulting heterodimeric species was tested in the TNFreceptor assay and found to have an IC50 for neutralising TNF ofapproximately 18 nM. Next, the receptor assay was repeated with aconstant concentration of heterodimer (18 nM) and a dilution series ofMSA and HSA. The presence of HSA at a range of concentrations (up to 2mg/ml) did not cause a reduction in the ability of the dimer to inhibitTNFα. However, the addition of MSA caused a dose dependant reduction inthe ability of the dimer to inhibit TNFα (FIG. 20). This demonstratesthat MSA and TNFα compete for binding to the cys bonded TAR1-5-19, MSA16dimer.

Data Summary

A summary of data obtained in the experiments set forth in the precedingexamples is set forth in Annex 4.

Example 13 Selection and Characterisation of dAbs for Binding to SerumAlbumin from a Range of Species

dAbs against human serum albumin, mouse serum albumin and porcine serumalbumin were selected as previously described for the anti-MSA dAbsexcept for the following modifications to the protocol: The phagelibraries of synthetic V_(H) domains were the libraries 4G and 6G, whichare based on a human V_(H)3 comprising the DP47 germ line gene and theJ_(H)4 segment for the VH and a human V_(κ)1 comprising the DPK9 germline gene and the J_(κ)1 segment for the V_(κ). The libraries comprise1×10¹⁰ individual clones. A subset of the V_(H) and V_(κ) libraries hadbeen preselected for binding to generic ligands protein A and protein Lrespectively so that the majority of clones in the unselected librarieswere functional. The sizes of the libraries shown above correspond tothe sizes after preselection.

Two or three rounds of selection were performed on mouse, porcine andhuman serum albumin using subsets of the V_(H) and V_(κ) librariesseparately. For each selection, antigen was either (i) coated onimmunotube (nunc) in 4 ml of PBS at a concentration of 100 μg/ml, or(ii) biotinylated and then used for soluble selection followed bycapture on streptavidin beads or neutravidin beads. In each case, afterthe second or third round of selection, DNA from the selection wascloned into an expression vector for production of soluble dAb, andindividual colonies were picked. Soluble dAb fragments were produced asdescribed for scFv fragments by Harrison et al (Methods Enzymol. 1996;267:83-109) and for each selection, 96 soluble clones were tested forbinding to a range of serum albumins.

Screening of clones for binding to serum albumins from a range ofspecies was done using a Biacore surface plasmon resonance instrument(Biacore AB). A CM-5 Biacore chip was coated with serum albumin fromdifferent species at high density on each of flow cells 2 to 4. dAbswhich exhibited binding to one or more serum albumins of interest weresequenced and expressed at a 50 ml scale, purified on protein L and thenscreened at a known concentration for binding to a panel of serumalbumins on a CM-5 Biacore chip coated with a low density of serumalbumin on flow cells 2 to 4. Several dAbs which bind serum albumin froma range of different species were found, with the preferred candidatesbeing listed, along with their binding profiles, in Table 7.

TABLE 7 HSA RSA MSA Cyno (affinity if (affinity if (affinity if(affinity if measured) measured) measured) measured) DOM7h-9 Binds bindsbinds binds 200 nM DOM7h-10 binds ND ND ND DOM7h-11 binds binds bindsbinds DOM7h-12 binds ND binds binds DOM7h-13 binds binds binds DOM7h-14Binds binds Binds Binds 38 nM 27 nM 123 nM

In this experiment, we have therefore isolated dAbs that bind HSA andalbumin from one or more of a range of non-human species. For example,we found dAbs that bind (i) human and mouse, (ii) human and cynomolgus,(iii) human and rat and (iv) human, mouse, rat and cyno albumin.

Example 14 Determination of the Serum Half-Life in Rat and CynomolgusMonkey of Serum Albumin Binding dAb/HA Epitope Tag or dAb/Myc EpitopeTag Fusion Proteins and Determination of Serum Half Life

Anti-cynomolgus serum albumin dAbs were expressed with C-terminal HA ormyc tags in the periplasm of E. coli and purified using batch absorptionto protein L-agarose affinity resin (Affitech, Norway) for Vk dAbs andbatch absorption to protein A affinity resin for VH dAbs, followed byelution with glycine at pH 2.0. In order to determine serum half life,groups of 3 cynomolgus macaques were given a single i.v. injection at2.5 mg/Kg of DOM7h-9, DOM7h-11 or DOM7h-14. Blood samples were obtainedby serial bleeds from a femoral vein or artery over a 21 day period andserum prepared from each sample. Serum samples were analysed by sandwichELISA using goat anti-HA (Abcam, Cambridge UK) or goat anti myc (Abcam,Cambridge UK) coated on an ELISA plate, followed by detection withprotein L-HRP. Standard curves of known concentrations of dAb were setup in the presence of cynomolgus serum at the same concentration as forthe experimental samples to ensure comparability with the test samples.Fitting a double exponential Modelling with a 2 compartment model (usingkaleidograph software (Synergy software, PA, USA)) was used to calculatet1/2β, see Table 8.

Anti-rat serum albumin dAbs were expressed with C-terminal HA or myctags in the periplasm of E. coli and purified using batch absorption toprotein L-agarose affinity resin (Affitech, Norway) followed by elutionwith glycine at pH 2.0. dAbs were then labelled with ³H using thefollowing method: One vial per protein was prepared: 300 μL of NSP wasdispensed into the vial and the solvent removed under a gentle stream ofnitrogen at ≦30° C. The residue was then re-suspended in DMSO (100 μL).An aliquot of protein solution (2.5 mL) was added to the DMSO solutionand the mixture incubated for 60 minutes at room temperature. Exactly2.5 ml of the solution was then be loaded onto a pre-equilibrated PD10column (pre-equilibrated with 25 mL Phosphate buffered saline, PBS) andthe eluate discarded. Phosphate buffered saline (PBS, 3.5 mL) will beadded and the eluate collected. This provided a labelled proteinsolution at approximately 2 mg/mL. The specific activity of the materialwas determined and conditional on efficient labelling, the solution wasused immediately or stored at −20° C. until required.

In order to determine serum half life, groups of 4 rats were given asingle i.v. injection at 2.5 mg/Kg of DOM7h-9, DOM7h-11, DOM7h-13 orDOM7h-14. Blood samples were obtained from a tail vein over a 7 dayperiod and plasma prepared. Levels of ³H were determined by liquidscintillation counting and concentration of labelled protein in eachsample calculated according to the known specific activity of theprotein administered at the start of the experiment. Fitting a doubleexponential Modelling with a 2 compartment model (using kaleidographsoftware (Synergy software, PA, USA)) was used to calculate t1/2β, seeTable 8.

TABLE 8 Agent Scaffold t½β (cyno) t½β (rat) DOM7h-9 V_(κ) 3.8 days 66hours DOM7h-11 V_(κ) 5.2 days 61 hours DOM7h-13 V_(κ) not tested 73hours DOM7h-14 V_(κ) 6.8 days 56 hours DOM7r-3 V_(κ) 53 hours DOM7r-16V_(κ) 43 hours DOM7h-9 V_(κ) 3.8 days 66 hours DOM7h-11 V_(κ) 5.2 days61 hours DOM7h-13 V_(κ) not tested 73 hours DOM7h-14 V_(κ) 6.8 days 56hours DOM7r-3 V_(κ) 53 hours DOM7r-16 V_(κ) 43 hours

The half life of albumin in rat and cynomolgus monkey is 53 hours(determined experimentally) and 7-8 days (estimated) respectively. Itcan be seen from Table 8 that the half life of dAbs DOM7r-3, DOM7h-9,DOM7h-11, DOM7h-13 and DOM7h-14 in rat approach or are substantially thesame as the half life of albumin in rat. Also, it can be seen that thatthe half life of dAbs DOM7h-11 and DOM7h-14 in cynomolgus approach orare substantially the same as the half life of albumin in cynomolgus.dAb DOM7h-14 has a half life in both rat and cynomolgus that issubstantially the same as the half life of albumin in both species.

Example 15. Epitope Mapping

The three domains of human serum albumin have previously been expressedin Pichia pastoris (Dockal Carter and Ruker (1999) J. Biol. Chem. 2000Feb. 4; 275(5):3042-50. We expressed the same domains using the Pichiapastoris pPICZaA vector and where required purified them to homogeneityon Mimetic Blue SA matrix (supplier: Prometic Biosciences) FIG. 21. Theidentification of the serum albumin domain bound by dAbs was assessed byone of two methods, immunoprecipitation of domain antibodies and bycompetition Biacore. Results are shown below in FIG. 22 and FIG. 23.

For immunoprecipitation assay, 1 ml of Pichia pastoris supernatantexpressing either HSA domain I, II or III was adjusted to pH7.4, andmixed with 1 μg dAb, and 10 μl of Protein A or Protein L agarose (forV_(H) or V_(κ) dAbs respectively). The mixture was mixed by inversionfor 1 hour to allow complex formation, then the agarose bound complexwas recovered by centrifugation at 13,000×g for 10 minutes, thesupernatant decanted, and the pelleted material washed once with PBS,and recovered by centrifugation. The beads were then resuspended inSDS-PAGE loading buffer containing dithiothreitol (DTT), heated to 70°C. for 10 minutes, then run on a 4-12% NuPAGE SDS-PAGE gels (supplier:Invitrogen), and stained with SimplyBlue safestain.

For competition Biacore assay, purified dAbs were made up to 1 μM inHBS-EP at pH7.4, or 1 μM in 50 mM citrate phosphate buffer, 150 mM NaCl,pH5.0, and where required, with 7 μM purified HSA domain. Biacore runswere carried out at a flow rate of 30 μl min over a CM5 chip surfacecoated with 500-1000 RU of human serum albumin, and a blank referencecell used to do baseline subtraction.

Table 9 provides a list of dAbs specific for human serum albumin and thedomain(s) of human serum albumin to which they map (as determined byimmunoprecipitation and/or Biacore):

TABLE 9 Clone H/K Mapped HSA domain DOM7h-1 K Domain II DOM7h-2 K NdDOM7h-6 K Nd DOM7h-7 K Nd DOM7h-8 K Domain II DOM7h-9 K Domain IIDOM7h-10 K Nd DOM7h-11 K Domain II DOM7h-12 K Domain II DOM7h-13 KDomain II DOM7h-14 K Domain II DOM7h-21 H Nd DOM7h-22 H Domain I + IIIDOM7h-23 H Nd DOM7h-24 H Nd DOM7h-25 H Nd DOM7h-26 H Nd DOM7h-27 HDomain III DOM7h-30 H Domain III DOM7h-31 H Nd Nd: not determined

In conclusion, the majority of dAbs bind to the 2nd domain of HSA andare therefore not expected to compete with binding of human serumalbumin to FcRn. Two dAbs (DOM7h-27 and DOM7h-30) bind to Domain III.

HSA RU HSA RU HSA domain binding at binding at dAb bound 1 μM pH 7.4 1μM pH 5.0 His in CDR DOM7h-1 II  600c 150 no DOM7h-3 NI  0 0 DOM7h-4 NI 0 0 DOM7h-8 II 1000  250 no DOM7h-9 II 150 0 CDR1 DOM7h-11 II 250 0CDR3 DOM7h-12 IIa  55 0 no DOM7h-13 II 300 40 2 in CDR3 DOM7h-14 II  200 no DOM7h-22 I + IIIb  100c 0 CDR2 DOM7h-27 III  50 0 no DOM7h-30 III320 35 no

Summary of results of epitope mapping of HSA binding AlbudAb™s (dAbswhich specifically binds serum albumin) and Biacore data at pH7.4 and5.0.

Example 16 Selecting dAbs In Vitro in the Presence of Metabolites

Albumin molecules accumulate the effects of exposure to other compoundsin serum during their lifetime of around 19 days. These effects includethe binding of numerous molecules that have affinity for albumin whichinclude but are preferably not limited to cysteine and glutathionecarried as mixed disulphides, vitamin B₆, δ-bilurubin, hemin, thyroxine,long and medium, chain fatty acids and glucose carried on ε-aminogroups. Also, metabolites such as acetaldehyde (a product of ethanolmetabolism in the liver), fatty acid metabolites, acyl glucuronide andmetabolites of bilirubin. In addition, many drugs such as warfarin,halothane, salicylate, benzodiazepines and others (reviewed in Fasano etal 2005, IUBMB Life)) and also 1-O-gemfibrozil-β-D-glucuronide bind toserum albumin.

Compounds found bound to serum albumin tend to bind at certain sites onthe albumin molecule, thereby potentially blocking these sites for thebinding of other molecules such as AlbudAbs™ (a dAb which specificallybinds serum albumin). The binding sites for many ligands has beenidentified, the main and most well characterised binding sites aretermed “Sudlow site 1” and “Sudlow site 2”. According to thisnomenclature, Site 1 is located in sub-domain IIA, and binds warfarinand other drugs which generally are bulky, heterocyclic anionicmolecules. Site 2 is located in sub domain IIIA, and binds aromaticcarboxylic acids with an extended conformation, with the negative chargetowards one end, such as the stereotypical site 2 ligand, ibuprofen.Secondary binding sites for both Warfarin and ibuprofen have beenidentified on domains II and I respectively. Other binding sites andsub-sites of these also exist, meaning that in the circulation, serumalbumin exists with a complex mixture of bound ligands, with affinitiesthat vary from 1×10⁻²M to 1×10⁻⁸M. Oleic acid for example binds to up 7sites on SA (J Mol Biol. 2001; 314:955-60).

Human serum albumin has been in crystallized complex with fatty acids(Petitpas I, Grune T, Bhattacharya A A, Curry S. Nat. Struct Biol.(1998) 5: 827-35). The binding sites for these molecules are situated inhydrophobic clefts around the SA surface, with an asymmetricdistribution, despite the near three-fold symmetry of the HSA molecule.Later, the use of various recombinant fragments of serum albumin hasaided more precise assignment of the contribution of the domains toformation of the binding sites (for example: Protein Sci (2000)9:1455-65; J Biol Chem. (1999) 274:29303-10). Displacement of boundligands from SA plays an important role in drug interactions, forexample the half life of warfarin is reduced as it is displaced from SAby ethanol (J Biol Chem. (2000) 275:38731-8). Other drugs affinity forSA is modified by the presence of other drugs in other binding sites.For example, diazepam binding to site 2 increases the affinity of site 1for tenoxicam, as a result of conformational changes on binding. Thissignificantly affects the pharmacokinetic properties (Fundam ClinPharmacol. (1989) 3:267-79).

Thus, for a SA binding AlbudAb™ (a dAb which specifically binds serumalbumin), it is desirable to select one that does not alter the bindingcharacteristics of serum albumin for drugs bound to SA. Additionally,where drug binding has been shown to alter the conformation of SA, it isdesirable to have an AlbudAb™(a dAb which specifically binds serumalbumin) that binds SA in both in the presence or absence of the drug.These approaches mean that it will be possible to identify an AlbudAb™(a dAb which specifically binds serum albumin) such that there are notsignificant positive or negative drug interactions with keypharmaceuticals. Therefore, this example describes a phage selection toidentify dAbs that bind serum albumin in the presence of compounds andmetabolites likely to be present bound to albumin in vivo. Phageselections are performed in the presence of one or several of themetabolites or compounds known to interact with serum albumin in vivo.These selections identify AlbudAb™s (a dAb which specifically bindsserum albumin) that will bind to serum albumin in a manner that isunlikely to be hindered by the presence of metabolites or othercompounds.

The phage libraries described in Example 1 are used as described inExample 1 for selection against albumin from one or more of a range ofspecies including human, cynomolgus monkey, rat and mouse. The albuminused as an antigen is different from that described in Example 1 in thatit will be preincubated overnight with ametabolite or compound at a10-100 fold higher concentration than the albumin itself. This caneither be with a single compound or metabolite, or with more than onecompound or metabolite. In particular, it can be in the presence ofcompounds occupying albumin site I or site II or both. Thisconcentration of metabolite is also present in the buffer used to coatthe immunotubes with antigen and in the buffers used during key steps ofthe selection. Steps where metabolites are present include the MPBSblocking buffer used to block the antigen coated immunotubes or thebiotinylated antigen (for solution selections) and also the buffer inwhich the phage library is blocked. In this way, when the blocked phageare added to the immunotube or biotinylated antigen, the concentrationof metabolite is maintained. Therefore, throughout the phases of theselection in which the phage that bind to albumin are selected,metabolites that may block certain sites on the albumin molecule in vivoare also present, competing with the phage for binding and biasing theselection in favour of those dAbs that bind sites on albumin differentfrom those blocked by metabolites.

In another set of selections, alternating rounds of selection againstserum albumin in the presence and absence of bound compounds ormetabolites are performed. This ensures that dAbs able to bind serumalbumin in both the presence and absence of bound compounds areselected. In both selection schemes, it is possible that dAbs that arecapable of displacing drug bound to serum albumin will be selected, andthis is screened for by measuring the ability of the AlbudAb™ (a dAbwhich specifically binds serum albumin) to displace SA bound drug. Suchassays are well established for small molecule drugs, and easily adaptedfor this purpose. A variety of methods well known in the art may be usedto determine the ability of an AlbudAb™ (a dAb which specifically bindsserum albumin) to displace SA bound drugs. These range from equilibriumdialysis, chromatographic methods on immobilised ligands or serumalbumin, through NMR analysis. The following example describes the useof the simplest equilibrium dialysis method. The other more technicallycomplex methods will give essentially the same information.

A solution of serum albumin is made at a defined concentration in aphysiological buffer, for example, 20 mM sodium phosphate buffer, 150 mMNaCl, pH7.4. The drug is made up in a similar buffer, and has beensynthesised such that it retains its original pharmacologicalproperties, but is radiolabelled, for example with tritium or ¹⁴C. Theserum albumin binding antibody fragment is made up at a definedconcentration in a similar buffer.

The serum albumin solution is placed in a series of tubes, andincreasing amount of AlbudAb™ (a dAb which specifically binds serumalbumin) is added, such that the concentration of serum albumin in eachtube is fixed (for example at 1% w/v, approx 150 μM), while the (a dAbwhich specifically binds serum albumin)™ concentration ranges from 0 to150 μM over the tube series. This comprises one experimental set.

A dialysis tube or container containing a fixed concentration of theradiolabelled ligand for each set is added to the tube. A concentrationrange from 0.2 to 10 mM may be suitable, depending on the ligand used,its affinity and solubility.

The cut-off size of the membrane used for dialysis should be such thatthe serum albumin and AlbudAb™ (a dAb which specifically binds serumalbumin) do not diffuse through, but the radiolabelled ligand candiffuse freely. A cut off size of 3.5 Kda is sufficient for thispurpose.

The mixture is stirred at a fixed temperature, for example 37° C., for afixed period of time, to allow equilibrium of the radiolabelled drugbetween both compartments, for example, 5 hours. After this time,equilibrium should be attained which is influenced by the ability of theAlbudAb™ (a dAb which specifically binds serum albumin) binding theserum albumin to inhibit drug binding.

Both compartments are samples, and the radioactivity counted, using ascintillation counter. The concentration of albumin bound ligand can bedetermined by the difference in counts between the two compartments. Thestoichiometric binding constant K′ can be calculated from theequilibrium concentration of bound ligand, b, free ligand, c, andalbumin, p, in accordance with the equation K′=b/c(p−b). This assumesthe binding of 1 molecule of ligand to one molecule of serum albumin.

Binding data can then be measured using a Scatchard plot in accordancewith the equation r/c=nk−rk, where r is the fraction of albumin to whichligand is bound (i.e. b/p, and n is the number of binding sites peralbumin molecule, and k is the site association constant. Values of nand k can be determined from plots of r/c against r.

Where the binding of an AlbudAb™ (a dAb which specifically binds serumalbumin) blocks radiolabelled ligand binding, this will affect both thestoichiometric binding constant of the ligand, and also the apparentnumber of binding sites for the ligand. It may be predicted that as theAlbudAb™ (a dAb which specifically binds serum albumin) will bind at onedefined site on the surface of serum albumin, and some ligands have morethan one binding site on serum albumin, that not all binding sites willbe blocked. In the situation where The AlbudAb™ (a dAb whichspecifically binds serum albumin) specifically binds to drug complexedserum albumin and displaces it, and the drug has a low therapeutic indexand is serum bound, then a cut-off affinity for distinguishing betweenan AlbudAb™ (a dAb which specifically binds serum albumin) able todisplace serum albumin bound to the drug from an AlbudAb™ (a dAb whichspecifically binds serum albumin) not able to displace serum albumin todrug, would range from 10 nM to 100 nM. This method is exemplified inthe following paper: Livesey and Lund Biochem J. 204(1): 265-272 Bindingof branched-chain 2-oxo acids to bovine serum albumin.

Example 17: Generation of Dual-Specific Ligand Comprising a SerumAlbumin-Binding CTLA-4 Non-Immunoglobulin Scaffold Via CDR Grafting

The CDR domains of dAb7h14 are used to construct a cytotoxicT-lymphocyte associated antigen 4 (CTLA-4) non-immunoglobulin scaffoldpolypeptide that binds human serum albumin in the following manner. TheCDR1 (RASQWIGSQLS; SEQ ID NO.: ______), CDR2 (WRSSLQS; SEQ ID NO.:______), and CDR3 (AQGAALPRT; SEQ ID NO.: ______) sequences of dAb7h14are grafted into a soluble truncated mutant of CTLA-4 comprising theCTLA-4 V-like domain (as described in WO 99/45110; optionally, anengineered form of CTLA-4, e.g., in which A2 and A3 domains are deleted)in replacement of native CTLA-4 amino acid residues corresponding toCDR1 (SPGKATE; SEQ ID NO.: ______) within the S1-S2 loop (the BC loop),CDR2 (YMMGNELTF; SEQ ID NO.: ______), and CDR3 (LMYPPPYYL; SEQ ID NO.:______) within the S5-S6 loop, respectively (for details of the CTLA-4scaffold composition and/or structure refer to WO 00/60070; WO 99/45110;Metzler et al. Nat. Struct. Biol. 4: 527-53; and Nuttall et al. ProteinsStruct. Funct. Genet. 36:217-27, all incorporated herein by reference intheir entirety). Expression of this CLTA-4-derived polypeptide in apGC-, pPOW-based, or other art-recognized expression system isperformed, with the anticipated production of predominantly monomericsoluble protein. Protein solubility of this CTLA-4-derived polypeptideis examined, and is anticipated to be superior to native extracellularCTLA-4 polypeptide. ELISA analysis is used to examine whether purifiedmonomeric polypeptide specifically binds human serum albumin compared tonon-specific antigens and compared to extracellular CTLA-4-derivedpolypeptides grafted with non-specific polypeptides (e.g., somatostatinsubstituted within the CDR1 loop structure). Real-time binding analysisby Biacore is performed to assess whether human serum albuminspecifically binds to immobilized CTLA-4-derived polypeptide comprisingthe anti-human serum albumin CDR domains of dAb7h14. (One of skill inthe art will recognize that binding affinity can be assessed using anyappropriate method, including, e.g., precipitation of labeled humanserum albumin, competitive Biacore assay, etc.) Optionally, expressionof the CTLA-4 anti-human serum albumin polypeptide is enhanced viaadjustment of the coding sequence using splice overlap PCR toincorporate codons preferential for E. coli expression. If no or lowhuman serum albumin affinity (e.g., Kd values in the μM range or higher)is detected, at least one of a number of strategies is employed toimprove the human serum albumin binding properties of the CDR-graftedCTLA-4 polypeptide, including any of the following methods thatcontribute to binding affinity.

Human serum albumin binding of CDR-grafted CTLA-4 polypeptide(s)presenting dAb7h14 CDRs is optimized via mutagenesis, optionally incombination with parallel and/or iterative selection methods asdescribed below and/or as otherwise known in the art. CTLA-4 scaffoldpolypeptide domains surrounding grafted dAb7h14 CDR polypeptidesequences are subjected to randomized and/or NNK mutagenesis, performedas described infra. Such mutagenesis is performed within the CTLA-4polypeptide sequence upon non-CDR amino acid residues, for the purposeof creating new or improved human serum albumin-binding polypeptides.Optionally, dAb7h14 CDR polypeptide domains presented within theCDR-grafted CTLA-4 polypeptide are subjected to mutagenesis via, e.g.,random mutagenesis, NNK mutagenesis, look-through mutagenesis and/orother art-recognized method. PCR is optionally used to perform suchmethods of mutagenesis, resulting in the generation of sequencediversity across targeted sequences within the CDR-grafted CTLA-4polypeptides. Such approaches are similar to those described infra fordAb library generation. In addition to random and/or look-throughmethods of mutagenesis, directed mutagenesis of targeted amino acidresidues is employed where structural information establishes specificamino acid residues to be critical to binding of human serum albumin.

CTLA-4 polypeptides comprising grafted dAb7h14 CDR sequences engineeredas described above are subjected to parallel and/or iterative selectionmethods to identify those CTLA-4 polypeptides that are optimized forhuman serum albumin binding. For example, following production of alibrary of dAb7h14 CDR-grafted CTLA-4 polypeptide sequences, thislibrary of such polypeptides is displayed on phage and subjected tomultiple rounds of selection requiring serum albumin binding and/orproliferation, as is described infra for selection of serumalbumin-binding dAbs from libraries of dAbs. Optionally, selection isperformed against serum albumin immobilized on immunotubes or againstbiotinlyated serum albumin in solution. Optionally, binding affinity isdetermined using surface plasmon resonance (SPR) and the Biacore(Karlsson et al., 1991), using a Biacore system (Uppsala, Sweden), withfully optimized CTLA-4-derived polypeptides ideally achieving humanserum albumin binding affinity Kd values in the nM range or better.

Following identification of CTLA-4-derived polypeptides that bind humanserum albumin, such polypeptides are then used to generate dual-specificligand compositions by any of the methods described infra.

Example 18: Generation of Dual-Specific Ligand Comprising a SerumAlbumin-Binding CTLA-4 Non-Immunoglobulin Scaffold Via Selection ofSerum Albumin Binding Moieties

A soluble truncated mutant of CTLA-4 comprising the native CTLA-4 V-likedomain (as described in WO 99/45110; optionally, an engineered form ofCTLA-4, e.g., in which A2 and A3 domains are deleted) and which has beenengineered to contain regions(s) of variability, are displayed in alibrary and subjected to selection and, optionally, affinity maturationtechniques in order to produce human serum albumin-binding CTLA-4non-immunoglobulin scaffold molecules for use in the ligands of theinvention.

Expression of this CLTA-4-derived polypeptide in a pGC-, pPOW-based, orother art-recognized expression system is performed. Protein solubilityof this CTLA-4-derived polypeptide is examined, and mutagenesis isperformed to enhance solubility of CTLA-4-derived polypeptide(s)relative to that of a native extracellular CTLA-4 polypeptide. ELISAanalysis is used to examine whether purified monomeric polypeptideoptionally specifically binds human serum albumin compared tonon-specific single varaible domains comprising a CTLA-4 derivedscaffold, and compared to extracellular CTLA-4-derived polypeptidesgrafted with non-specific polypeptides (e.g., CTLA-4 polypeptide withsomatostatin substituted within the CDR1 loop structure). Real-timebinding analysis by Biacore is performed to assess whether human serumalbumin specifically binds to immobilized CTLA-4-derived polypeptide.Optionally, expression of the CTLA-4 anti-human serum albuminpolypeptide is enhanced via adjustment of the coding sequence usingsplice overlap PCR to incorporate codons preferential for E. coliexpression. Following detection of no or low binding affinity (e.g., Kdvalues in the μM range or higher) of a CTLA-4 polypeptide for humanserum albumin, at least one of a number of strategies is employed toimpart human serum albumin binding properties to the CTLA-4 polypeptide,including one or more of the following methods that contribute tobinding affinity.

Human serum albumin binding of CTLA-4 scaffold polypeptide(s) isachieved and optimized via mutagenic methods, optionally in combinationwith parallel and/or iterative selection methods as described belowand/or as otherwise known in the art. CTLA-4 polypeptide domains aresubjected to randomized and/or NNK mutagenesis, performed as describedinfra. Such mutagenesis is performed upon the entirety of the CTLA-4polypeptide or upon specific sequences within the CTLA-4 polypeptide,optionally targeting CDR-corresponding amino acids (e.g., CDR1 and/orCDR3 sequences are randomized, and resulting polypeptides are subjectedto selection, e.g., as described in Example 6 of WO 99/45110).Optionally, specific amino acid residues determined or predicted to bestructurally important to CDR-like loop presentation are targeted formutagenesis. Mutagenesis, especially randomized mutagenesis, isperformed in order to evolve new or improved human serum albumin-bindingpolypeptides. PCR is optionally used to perform such methods ofmutagenesis, resulting in the generation of sequence diversity acrosstargeted sequences within the CTLA-4 polypeptides. (Such approaches aresimilar to those described infra for dAb library generation.) Inaddition to random methods of mutagenesis, directed mutagenesis oftargeted amino acid residues is employed where structural informationestablishes specific amino acid residues of CTLA-4 polypeptides to becritical to binding of human serum albumin.

CTLA-4 polypeptides engineered as described above are subjected toparallel and/or iterative selection methods to identify those CTLA-4polypeptides that are optimized for human serum albumin binding. Forexample, following production of a library of mutagenized CTLA-4polypeptide sequences, said library of polypeptides is displayed onphage and subjected to multiple rounds of selection requiring serumalbumin binding and/or proliferation, as is described infra forselection of serum albumin-binding dAbs from libraries of dAbs.Optionally, selection is performed against serum albumin immobilized onimmunotubes or against biotinlyated serum albumin in solution.Optionally, binding affinity is determined using surface plasmonresonance (SPR) and the Biacore (Karlsson et al., 1991), using a Biacoresystem (Uppsala, Sweden), with fully optimized CTLA-4-derivedpolypeptides ideally achieving human serum albumin binding affinity Kdvalues in the nM range or better.

Following identification of CTLA-4 polypeptides that bind human serumalbumin, such polypeptides are then used to generate dual-specificligand compositions by any of the methods described infra.

CTLA-4 V-Like Domains

CTLA-4 is an example of a non-immunoglobulin ligand that binds to aspecific binding partner and also comprises V-like domains. These V-likedomains are distinguished from those of antibodies or T-cell receptorsbecause they have no propensity to join together into Fv-type molecules.Such a non-immunoglobulin ligand provides an alternative framework forthe development of novel binding moieties with high affinities fortarget molecules. Single domain V-like binding molecules derived fromCTLA-4 which are soluble are therefore desirable.

Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is involved inT-cell regulation during the immune response. CTLA-4 is a 44 Kdahomodimer expressed primarily and transiently on the surface ofactivated T-cells, where it interacts with CD80 and CD86 surfaceantigens on antigen presenting cells to effect regulation of the immuneresponse (Waterhouse et al. 1996 Immunol Rev 153: 183-207, van der Merweet al. 1997 J Exp Med 185: 393-403). Each CTLA-4 monomeric subunitconsists of an N-terminal extracellular domain, transmembrane region andC-terminal intracellular domain. The extracellular domain comprises anN-terminal V-like domain (VLD; of approximately 14 Kda predictedmolecular weight by homology to the immunoglobulin superfamily) and astalk of about 10 residues connecting the VLD to the transmembraneregion. The VLD comprises surface loops corresponding to CDR-1, CDR-2and CDR-3 of an antibody V-domain (Metzler et al. 1997 Nat Struct Biol4: 527-531). Recent structural and mutational studies on CTLA-4 indicatethat binding to CD80 and CD86 occurs via the VLD surface formed fromA′GFCC′ V-like beta-strands and also from the highly conserved MYPPPYsequence in the CDR3-like surface loop (Peach et al. 1994 J Exp Med 180:2049-2058; Morton et al. 1996 J. Immunol. 156: 1047-1054; Metzler et al.1997 Nat Struct Biol 4: 527-531). Dimerisation between CTLA-4 monomersoccurs through a disulphide bond between cysteine residues (CYS120) inthe two stalks, which results in tethering of the two extracellulardomains, but without any apparent direct association between V-likedomains (Metzler et al. 1997 Nat Struct Biol 4: 527-531).

Replacement of CDR loop structures within the VLDs has previously beenshown to result in the production of monomeric, correctly foldedmolecules with altered binding specificities and improved solubility.Accordingly, in certain embodiments, a binding moiety comprising atleast one monomeric V-like domain (VLD) derived from CTLA-4 isgenerated, wherein the at least one monomeric V-like domain ischaracterized in that at least one CDR loop structure or part thereof ismodified or replaced such that the solubility of the modified VLD isimproved when compared with the unmodified VLD.

In certain embodiments, at least one CDR loop structure or part thereofis modified or replaced such that (i) the size of the CDR loop structureis increased when compared with corresponding CDR loop structure in theunmodified VLD; and/or (ii) the modification or replacement results inthe formation of a disulphide bond within or between one or more of theCDR loop structures.

In certain embodiments, the present invention provides a binding moietycomprising at least one monomeric V-like domain (VLD) derived fromCTLA-4, the at least one monomeric V-like domain being characterized inthat at least one CDR loop structure or part thereof is modified orreplaced such that (i) the size of the CDR loop structure is alteredwhen compared with corresponding CDR loop structure in the unmodifiedVLD; and/or (ii) the modification or replacement results in theformation of a disulphide bond within or between one or more of the CDRloop structures.

In certain embodiments, the size of the CDR loop structure is increasedby at least two, more preferably at least three, more preferably atleast six and more preferably at least nine amino acid residues. Infurther embodiments, the modified binding moiety of the invention alsoexhibits an altered binding affinity or specificity when compared withthe unmodified binding moiety. Preferably, the effect of replacing ormodifying the CDR loop structure is to reduce or abolish the affinity ofthe VLD to one or more natural ligands of the unmodified VLD.Preferably, the effect of replacing or modifying the CDR loop structureis also to change the binding specificity of the VLD (e.g., to produce acomposition that binds human serum albumin). Thus, it is preferred thatthe modified VLD binds to a specific binding partner (e.g., human serumalbumin) that is different to that of the unmodified VLD.

The phrase “VLD” is intended to refer to a domain which has similarstructural features to the variable heavy (VH) or variable light (VL)antibody. These similar structural features include CDR loop structures.

As used herein, the term “CDR loop structures” refers to surfacepolypeptide loop structures or regions like the complementaritydetermining regions in antibody V-domains.

It will be appreciated that the CTLA-4-derived binding moieties of thepresent invention may be coupled together, either chemically orgenetically, to form multivalent or multifunctional reagents. Forexample, the addition of C-terminal tails, such as in the native CTLA-4with Cys'20, will result in a dimer. The binding moieties of the presentinvention may also be coupled to other molecules for variousformulations, including those comprising dual specific ligands. Forexample, the CTLA-4 VLDs may comprise a C-terminal polypeptide tail ormay be coupled to streptavidin or biotin. The CTLA-4 VLDs may also becoupled to radioisotopes, dye markers or other imaging reagents for invivo detection and/or localization of cancers, blood clots, etc. TheCTLA-4 VLDs may also be immobilized by coupling onto insoluble devicesand platforms for diagnostic and biosensor applications.

In certain embodiments of the present invention, the extracellularCTLA-4 V-like domain is used. One or more surface loops of the CTLA-4V-like domain and preferably the CDR1, CDR2 or CDR3 loop structures arereplaced with a polypeptide which has a binding affinity for serumalbumin (e.g., CDR domains of dAb7h14 and sequences derived therefrom,as exemplified infra). It will be appreciated that these CTLA-4 VLDs maybe polyspecific, having affinities directed by both their naturalsurfaces and modified polypeptide loops.

One or more of the CDR loop structures of the CTLA-4 VLD can be replacedwith one or more CDR loop structures derived from an antibody. Theantibody may be derived from any species. In a preferred embodiment, theantibody is derived from a human, rat, mouse, camel, llama or shark. TheCDR1 and CDR3 loop structures may adopt non-canonical conformationswhich are extremely heterologous in length. The V-like domain may alsopossess a disulphide linkage interconnecting the CDR1 and CDR3 loopstructures (as found in some camel VHH antibodies) or the CDR2 and CDR3loop structures (as found in some llama VHH antibodies).

For in vivo applications it is preferable that VLDs are homologous tothe subject of treatment or diagnosis and that any possible xenoantigensare removed. Accordingly, it is preferred that VLD molecules for use inclinical applications are substantially homologous to naturallyoccurring human immunoglobulin superfamily members.

Serum albumin binding of CTLA-4 polypeptides (e.g., VLDs derived fromCTLA-4) can be optimized via selection of a binding moiety with anaffinity for serum albumin, e.g., comprising screening a library ofpolynucleotides for expression of a binding moiety with an affinity forserum albumin, wherein the polynucleotides have been subjected tomutagenesis which results in a modification or replacement in at leastone CDR loop structure in at least one VLD and wherein the solubility ofthe isolated modified VLD is improved when compared with the isolatedunmodified VLD.

It will be appreciated by those skilled in the art that within thecontext of such affinity screening method, any method of random ortargeted mutagenesis may be used to introduce modifications into theV-like domains. In a preferred embodiment, the mutagenesis is targetedmutagenesis. Optionally, the targeted mutagenesis involves replacementof at least one sequence within at least one CDR loop structure using,e.g., splice overlap or other PCR technology.

It will also be appreciated by those skilled in the art that thepolynucleotide library may contain sequences which encode VLDscomprising CDR loop structures which are substantially identical to CDRloop structures found in naturally occurring immunoglobulins and/orsequences which encode VLDs comprising non-naturally occurring CDR loopstructures. Optionally, the screening process involves displaying themodified V-like domains as gene III protein fusions on the surface ofbacteriophage particles.

The library may comprise bacteriophage vectors such as pHFA, fd-tet-dogor pFAB.5c containing the polynucleotides encoding the V-like domains.The screening process can also involve displaying the modified V-likedomains in a ribosomal display selection system.

The preferred CTLA-4-derived serum albumin binding molecules of thepresent invention provide the following advantages (i) use of a nativehuman protein obviates the need for subsequent humanization of therecombinant molecule, a step often required to protect against immunesystem response if used in human treatment; (ii) the domain is naturallymonomeric as described above (incorporation of residue Cys120 in aC-terminal tail results in production of a dimeric molecule); and (iii)structural modifications have resulted in improved E. coli expressionlevels.

Initial determination of native CTLA-4 structure allowed modeling andprediction of the regions corresponding to antibody CDR1, 2 and 3regions. It was hypothesized that such areas would be susceptible tomutation or substitution without substantial effect upon the molecularframework and hence would allow expression of a correctly foldedmolecule. The published structure of CTLA-4 (Metzler et al. 1997 NatStruct Biol 4: 527-531) showed these predictions to be accurate, despitethe unexpected separation of CDR1 from the ligand-binding site, and theextensive bending of CDR3 to form a planar surface contiguous with theligand binding face.

V-like domains provide a basic framework for constructing soluble,single domain molecules, where the binding specificity of the moleculemay be engineered by modification of the CDR loop structures. The basicframework residues of the V-like domain may be modified in accordancewith structural features present in camelid antibodies. The camel heavychain immunoglobulins differ from “conventional” antibody structures byconsisting of VHH chains, (Hamers-Casterman et al. 1993 Nature 363:446-448). Cammelid antibldies consist of two heavy chains, eachcomprising a V_(HH) domain. Several unique features allow theseantibodies to overcome the dual problems of solubility and inability topresent a sufficiently large antigen binding surface.

First, several non-conventional substitutions (predominantly hydrophobicto polar in nature) at exposed framework residues reduce the hydrophobicsurface, while maintaining the internal beta-sheet framework structure(Desmyter et al. 1996 Nat Struct Biol 3:803-811). Further, within thethree CDR loops several structural features compensate for the loss ofantigen binding-surface usually provided by the V_(L) domain. While theCDR2 loop does not differ extensively from other VH domains, the CDR1and CDR3 loops adopt non-canonical conformations which are extremelyheterologous in length. For example, the H1 loop may contain anywherebetween 2-8 residues compared to the usual five in Ig molecules.However, it is the CDR3 loop which exhibits greatest variation: in 17camel antibody sequences reported, the length of this region variesbetween 7 and 21 residues (Muyldermans et al. 1994 Protein Eng 7:1129-1135). Thirdly, many camelid VHH domains possess a disulphidelinkage interconnecting CDR1 and CDR3 in the case of camels andinterconnecting CDR1 and CDR2 in the case of llamas (Vu et al. 1997Molec. Immunol. 34: 1121-113). The function of this structural featureappears to be maintenance of loop stability and providing a morecontoured, as distinct from planar, loop conformation which both allowsbinding to pockets within the antigen and gives an increased surfacearea. However, not all camelid antibodies possess this disulphide bond,indicating that it is not an absolute structural requirement.

The present invention also relates to a method for generating andselecting single VLD molecules with novel binding affinities for targetmolecules (e.g., human serum albumin). This method involves theapplication of well known molecular evolution techniques toCTLA-4-derived polypeptides. The method may involve the production ofphage or ribosomal display libraries for screening large numbers ofmutated CTLA-4-derived polypeptides.

Filamentous fd-bacteriophage genomes are engineered such that the phagedisplay, on their surface, proteins such as the Ig-like proteins (scFv,Fabs) which are encoded by the DNA that is contained within the phage(Smith, 1985 Science 228: 1315-1317; Huse et al. 1989 Science 246:1275-81; McCafferty et al., 1990 Nature 348: 552-4; Hoogenboom et al.,1991 Nucleic Acids Res. 19: 4133-4137). Protein molecules can bedisplayed on the surface of Fd bacteriophage, covalently coupled tophage coat proteins encoded by gene III, or less commonly gene VIII.Insertion of antibody genes into the gene III coat protein givesexpression of 3-5 recombinant protein molecules per phage, situated atthe ends. In contrast, insertion of antibody genes into gene VIII hasthe potential to display about 2000 copies of the recombinant proteinper phage particle, however this is a multivalent system which couldmask the affinity of a single displayed protein. Fd phagemid vectors arealso used, since they can be easily switched from the display offunctional Ig-like fragments on the surface of fd-bacteriophage tosecreting soluble Ig-like fragments in E. coli. Phage-displayedrecombinant protein fusions with the N-terminus of the gene III coatprotein are made possible by an amber codon strategically positionedbetween the two protein genes. In amber suppressor strains of E. coli,the resulting Ig domain-gene III fusions become anchored in the phagecoat.

A selection process based on protein affinity can be applied to anyhigh-affinity binding reagents such as antibodies, antigens, receptorsand ligands (see, e.g., Winter and Milstein, 1991 Nature 349: 293-299,the entire contents of which are incorporated herein by reference).Thus, the selection of the highest affinity binding protein displayed onbacteriophage is coupled to the recovery of the gene encoding thatprotein. Ig- or non-Ig scaffold-displaying phage can be affinityselected by binding to cognate binding partners covalently coupled tobeads or adsorbed to plastic surfaces in a manner similar to ELISA orsolid phase radioimmunoassays. While almost any plastic surface willadsorb protein antigens, some commercial products are especiallyformulated for this purpose, such as Nunc Immunotubes.

Ribosomal display libraries involve polypeptides synthesized de novo incell-free translation systems and displayed on the surface of ribosomesfor selection purposes (Hanes and Pluckthun, 1997 Proc. Natl. Acad. Sci.USA. 94: 4937-4942; He and Taussig, 1997 Nucl. Acids Res. 25:5132-5134). The “cell-free translation system” comprises ribosomes,soluble enzymes required for protein synthesis (usually from the samecell as the ribosomes), transfer RNAs, adenosine triphosphate, guanosinetriphosphate, a ribonucleoside triphosphate regenerating system (such asphosphoenol pyruvate and pyruvate kinase), and the salts and bufferrequired to synthesize a protein encoded by an exogenous mRNA. Thetranslation of polypeptides can be made to occur under conditions whichmaintain intact polysomes, i.e. where ribosomes, mRNA molecule andtranslated polypeptides are associated in a single complex. Thiseffectively leads to “ribosome display” of the translated polypeptide.For selection, the translated polypeptides, in association with thecorresponding ribosome complex, are mixed with a target (e.g., serumalbumin) molecule which is bound to a matrix (e.g., Dynabeads). Theribosomes displaying the translated polypeptides will bind the targetmolecule and these complexes can be selected and the mRNA re-amplifiedusing RT-PCR.

Although there are several alternative approaches to modify bindingmolecules, the general approach for all displayed proteins conforms to apattern in which individual binding reagents are selected from displaylibraries by affinity to their cognate ligand and/or receptor. The genesencoding these reagents are modified by any one or combination of anumber of in vivo and in vitro mutation strategies and constructed as anew gene pool for display and selection of the highest affinity bindingmolecules.

Assessment of Binding Affinities

In certain embodiments, the dual-specific ligands of the presentinvention, including component molecules thereof (e.g.,non-immunoglobulin molecules that bind human serum albumin) are assessedfor binding affinity to target protein (e.g., human serum albumin).Binding of target protein epitopes can be measured by conventionalantigen binding assays, such as ELISA, by fluorescence based techniques,including FRET, or by techniques such as surface plasmon resonance whichmeasure the mass of molecules. Specific binding of an antigen-bindingprotein to an antigen or epitope can be determined by a suitable assay,including, for example, Scatchard analysis and/or competitive bindingassays, such as radioimmunoassays (RIA), enzyme immunoassays such asELISA and sandwich competition assays, and the different variantsthereof.

Binding affinity is preferably determined using surface plasmonresonance (SPR) and the Biacore (Karlsson et al., 1991), using a Biacoresystem (Uppsala, Sweden). The Biacore system uses surface plasmonresonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23: 1; Morton andMyszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecularinteractions in real time, and uses surface plasmon resonance which candetect changes in the resonance angle of light at the surface of a thingold film on a glass support as a result of changes in the refrativeindex of the surface up to 300 nm away. Biacore analysis convenientlygenerates association rate constants, dissociation rate constants,equilibrium dissociation constants, and affinity constants. Bindingaffinity is obtained by assessing the association and dissociation rateconstants using a Biacore surface plasmon resonance system (Biacore,Inc.). A biosensor chip is activated for covalent coupling of the targetaccording to the manufacturer's (Biacore) instructions. The target isthen diluted and injected over the chip to obtain a signal in responseunits (RU) of immobilized material. Since the signal in RU isproportional to the mass of immobilized material, this represents arange of immobilized target densities on the matrix. Dissociation dataare fit to a one-site model to obtain k_(off)+/−s.d. (standard deviationof measurements). Pseudo-first order rate constant (Kd's) are calculatedfor each association curve, and plotted as a function of proteinconcentration to obtain k_(on)+/−s.e. (standard error of fit).Equilibrium dissociation constants for binding, Kd's, are calculatedfrom SPR measurements as k_(off)/k_(on).

As described by Phizicky and Field in Microb. Rev. (1995) 59: 114-115, asuitable antigen, such as HSA, is immobilized on a dextran polymer, anda solution containing a ligand for HSA, such as a single variabledomain, flows through a cell, contacting the immobilized HSA. The singlevariable domain retained by immobilized HSA alters the resonance angleof impinging light, resulting in a change in refractive index broughtabout by increased amounts of protein, i.e. the single variable domain,near the dextran polymer. Since all proteins have the same refractiveindex and since there is a linear correlation between resonance angleshift and protein concentration near the surface, changes in the proteinconcentration at the surface due to protein/protein binding can bemeasured, see Phizicky and Field, supra. To determine a bindingconstant, the increase in resonance units is measured as a function oftime by passing a solution of single variable domain protein past theimmobilized ligand (HSA) until the RU values stabilize, then thedecrease in RU is measured as a function of time with buffer lacking thesingle variable domain. This procedure is repeated at several differentconcentrations of single variable domain protein. Detailed theoreticalbackground and procedures are described by R. Karlsson, et. al. (991) J.Immunol Methods, 145, 229.

The instrument software produces an equilibrium dissociation constant(Kd) as described above. An equilibrium dissociation constant determinedthrough the use of Surface plasmon resonance (SPR) is described in U.S.Pat. No. 5,573,957, as being based on a table of dR_(A)/dt and R_(A)values, where R in this example is the HSA/single variable domaincomplex as measured by the Biacore in resonance units and where dR/dt isthe rate of formation of HSA/single variable domain complexes, i.e. thederivative of the binding curve; plotting the graph dR_(A)/dt vs. R_(A)for several different concentrations of single variable domain, andsubsequently plotting the slopes of these lines vs. the concentration ofsingle variable domain, the slope of this second graph being theassociation rate constant (M⁻¹, s⁻¹). The Dissociation Rate Constant orthe rate at which the HSA and the single variable domain release fromeach other can be determined utilizing the dissociation curve generatedon the Biacore. By plotting and determining the slope of the log of thedrop in response vs. time curve, the dissociation rate constant can bemeasured. The Equilibrium dissociation constant Kd=Dissociation RateConstant/association rate constant.

A ligand according to any aspect of the present invention, includes aligand having or consisting of at least one single variable domain, inthe form of a monomer single variable domain or in the form of multiplesingle variable domains, i.e. a multimer. The ligand can be modified tocontain additional moieties, such as a fusion protein, or a conjugate.Such a multimeric ligand, e.g., in the form of a dual-specific ligand,and/or such a ligand comprising or consisting of a single variabledomain, i.e. a dAb monomer useful in constructing such a multimericligand, may advantageously dissociate from their cognate target(s) witha Kd of 300 nM or less, 300 nM to 5 pM (i.e., 3×10⁻⁷ to 5×10⁻¹²M),preferably 50 nM to 20 pM, or 5 nM to 200 pM or 1 nM to 100 pM, 1×10⁻⁷ Mor less, 1×10⁻⁸ M or less, 1×10⁻⁹ M or less, 1×10⁻¹⁰ M or less, 1×10⁻¹¹Mor less; and/or a K_(off) rate constant ranging from 5×10⁻¹ to 1×10⁻⁷S⁻¹, preferably 1×10⁻⁶ to 1×10⁻⁸ S⁻¹, preferably 1×10⁻² to 1×10⁻⁶ S⁻¹,or 5×10⁻³ to 1×10⁻⁵ S⁻¹, or 5×10⁻¹ S⁻¹ or less, or 1×10⁻² S⁻¹ or less,or 1×10⁻³ S⁻¹ or less, or 1×10⁻⁴ S⁻¹ or less, or 1×10⁻⁵ S⁻¹ or less, or1×10⁻⁶ S⁻¹ or less as determined, for example, by surface plasmonresonance. The Kd rate constant is defined as K_(off)/K_(on).Preferably, a single variable domain will specifically bind a targetantigen or epitope with an affinity of less than 500 nM, preferably lessthan 200 nM, and more preferably less than 10 nM, such as less than 500pM

Lipocalins Example 19: Generation of Dual-Specific Ligand Comprising aSerum Albumin-Binding Lipocalin Non-Immunoglobulin Scaffold ViaSelection of Serum Albumin Binding Moieties

The bilin-binding protein (BBP), a lipocalin derived from Pierisbrassicae can be reshaped by combinatorial protein design such that itrecognizes human serum albumin. To this end, native BBP is subjected tolibrary selection and, optionally, affinity maturation in order toproduce human serum albumin-binding BBP molecules for use indual-specific ligands of the invention.

The capability of a native BBP to bind human serum albumin is initiallyascertained via Biacore assay, as described infra for CTLA-4-derivedpolypeptides. (One of skill in the art will recognize that bindingaffinity can be assessed using any appropriate method, including, e.g.,precipitation of labeled human serum albumin, competitive Biacore assay,etc.) Following detection of no or low binding affinity (e.g., Kd valuesin the μM range or higher) of BBP for human serum albumin, at least oneof a number of strategies are employed to impart human serum albuminbinding properties to BBP, including one or more of the followingmethods that contribute to binding affinity.

Human serum albumin binding of BBP and BBP-derived polypeptide(s) isachieved and optimized via mutagenic methods, optionally in combinationwith parallel and/or iterative selection methods as described belowand/or as otherwise known in the art. BBP polypeptide domains aresubjected to randomized and/or NNK mutagenesis, performed as describedinfra. Such mutagenesis is performed upon the entirety of the BBP (orBBP-derived) polypeptide and/or is performed upon specific sequenceswithin the BBP polypeptide, including 16 amino acid residues identifiedto reside at the center of the native BBP binding site, which is formedby four loops on top of an eight-stranded beta-barrel (Beste et al. 1999Proc. Natl Acad. Sci. USA 96: 1898-903). Optionally, such mutagenesisprocedures are randomized in order to evolve new or improved human serumalbumin-binding polypeptides; and multiple rounds of mutagenesis may beperformed during the process of creating a BBP that optimally binds tohuman serum albumin. PCR is optionally used to perform such methods ofmutagenesis, resulting in the generation of sequence diversity acrosstargeted sequences within the BBP (or BBP-derived) polypeptides. (Suchapproaches are similar to those described infra for dAb librarygeneration.) In addition to random methods of mutagenesis, directedmutagenesis of targeted amino acid residues is employed where structuralinformation establishes specific amino acid residues of BBP (orBBP-derived) polypeptides to be critical to binding of human serumalbumin.

BBP (or BBP-derived) polypeptides engineered as described above aresubjected to parallel and/or iterative selection methods to identifythose BBP polypeptides that are optimized for human serum albuminbinding. For example, following production of a library of mutagenizedBBP polypeptide sequences, said library of polypeptides is displayed onphage and subjected to multiple rounds of selection requiring serumalbumin binding and/or proliferation, as is described infra forselection of serum albumin-binding dAbs from libraries of dAbs.Optionally, selection is performed against serum albumin immobilized onimmunotubes or against biotinlyated serum albumin in solution.Optionally, binding affinity is determined using surface plasmonresonance (SPR) and the Biacore (Karlsson et al., 1991), using a Biacoresystem (Uppsala, Sweden), with fully optimized BBP-derived polypeptidesideally achieving human serum albumin binding affinity Kd values in thenM range or better.

Following identification of BBP polypeptides that bind human serumalbumin, such polypeptides are then used to generate dual-specificligand compositions by any of the methods described infra.

Lipocalin Scaffold Proteins

The lipocalins (Pervaiz and Brew, FASEB J. 1 (1987), 209-214) are afamily of small, often monomeric secretory proteins that have beenisolated from various organisms, and whose physiological role lies inthe storage or in the transport of different ligands as well as in morecomplex biological functions (Flower, Biochem. J. 318 (1996), 1-14). Thelipocalins exhibit relatively little mutual sequence similarity andtheir belonging to the same protein structural family was firstelucidated by X-ray structure analysis (Sawyer et al., Nature 327(1987), 659).

The first lipocalin of known spatial structure was the retinol-bindingprotein, Rbp, which effects the transport of water-insoluble vitamin Ain blood serum (Newcomer et al., EMBO J. 3 (1984), 1451-1454). Shortlythereafter, the tertiary structure of the bilin-binding protein, Bbp,from the butterfly Pieris brassicae was determined (Huber et al., J.Mol. Biol. 195 (1987), 423-434). The essential structural features ofthis class of proteins is illustrated in the spatial structure of thislipocalin. The central element in the folding architecture of thelipocalins is a cylindrical β-pleated sheet structure, a so-calledβ-barrel, which is made up of eight nearly circularly arrangedantiparallel β-strands.

This supersecondary structural element can also be viewed as a“sandwich”-arrangement of two four-stranded β-sheet structures.Additional structural elements are an extended segment at theamino-terminus of the polypeptide chain and an α-helix close to thecarboxy-terminus, which itself is followed by an extended segment. Theseadditional features are, however, not necessarily revealed in alllipocalins. For example, a significant part of the N-terminal segment ismissing in the epididymal retinoic acid-binding protein (Newcomer,Structure (1993) 1: 7-18). Additional peculiar structural elements arealso known, such as, for example, membrane anchors (Bishop and Weiner,Trends Biochem. Sci. (1996) 21: 127) which are only present in certainlipocalins.

The β-barrel is closed on one end by dense amino acid packing as well asby loop segments. On the other end, the β-barrel forms a binding pocketin which the respective ligand of the lipocalin is complexed. The eightneighboring antiparallel β-strands there are connected in a respectivepairwise fashion by hairpin bends in the polypeptide chain which,together with the adjacent amino acids which are still partially locatedin the region of the cylindrical β-pleated sheet structure, each form aloop element. The binding pocket for the ligands is formed by these intotal four peptide loops. In the case of Bbp, biliverdin IXy iscomplexed in this binding pocket. Another typical ligand for lipocalinsis vitamin A in the case of Rbp as well as β-lactoglobulin (Papiz etal., Nature 324 (1986), 383-385).

As described, for example, in U.S. Publication No. 20060058510, membersof the lipocalin family of polypeptides can be used to produce a classof molecules termed “anticalins” designed to recognize novel ligands viamutation of amino acids which are located in the region of the fourpeptide loops at the end of the cylindrical β-pleated sheet structure,and which are characterized in that they bind given ligands (e.g., humanserum albumin) with a determinable affinity.

Ligand-binding sites of the lipocalins are constructed more simply thanthose of immunoglobulins. Lipocalin polypeptides comprise only one ringof 8 antiparallel β-strands: the β-barrel. This cyclic β-pleated sheetstructure is conserved in the protein fold of the lipocalins. Thebinding site is formed in the entry region of the β-barrel by the fourpeptide loops, each of which connects two neighboring β-strands with oneanother. These peptide loops can vary significantly in their structurebetween the individual members of the lipocalin family.

To use a lipocalin polypeptide as a non-immunoglobulin scaffold, one ormore of the four peptide loops forming the ligand-binding site of alipocalin is subjected to mutagenesis, followed by choosing, i.e.selecting those protein variants (muteins), that exhibit the desiredbinding activity for a given ligand. The lipocalin muteins obtained inthis way have been termed “anticalins”.

The four peptide loops of the lipocalins which, during production ofanticalins, are modified in their sequence by mutagenesis, arecharacterized by those segments in the linear polypeptide sequence ofBBP comprising amino acid positions 28 to 45, 58 to 69, 86 to 99 and 114to 129 of Bbp. Each of these sequence segments begins before theC-terminus of one of the conserved β-strands at the open side of theβ-barrel, includes the actual peptide hairpin, and ends after theN-terminus of the likewise conserved β-strand which follows in thesequence.

Sequence alignments or structural superpositions allow the sequencepositions given for Bbp to be assigned to other lipocalins. For example,sequence alignments corresponding to the published alignment of Peitschand Boguski (New Biologist 2 (1990), 197-206) reveal that the fourpeptide loops of ApoD include the amino acid positions 28 to 44, 59 to70, 85 to 98 and 113 to 127. It is also possible to identify thecorresponding peptide loops in new lipocalins which are suitable formutagenesis in the same way.

In some cases, relatively weak sequence homology of the lipocalins mayprove to be problematic in the determination of the conserved β-strands.It is therefore crucial that the polypeptide sequence be capable offorming the cyclic β-pleated sheet structure made of 8 antiparallelβ-strands. This can be determined by employing methods of structuralanalysis such as protein crystallography or multidimensional nuclearmagnetic resonance spectroscopy.

In non-Bbp lipocalins, such as, for example, ApoD or Rbp, sequencesegments suitable for mutagenesis can easily be longer or shorter thanthat of Bbp based on the individually varying structure of the peptideloops. It can even be advantageous to additionally modify the length ofsequence segments by deletion or insertion of one or more amino acids.In certain embodiments, those amino acid positions corresponding tosequence positions 34 to 37, 58, 60, 69, 88, 90, 93, 95, 97, 114, 116,125, and 127 of Bbp are mutated. Correspondingly, in the case of ApoD,the sequence positions 34 to 37, 59, 61, 70, 87, 89, 92, 94, 96, 113,115, 123 and 125 are preferred for mutagenesis. However, for theproduction of anticalins, not all of the sequence positions listed abovehave to be subjected to mutagenesis.

Other lipocalins are also suitable as an underlying structure for theproduction of anticalins. Preferably, the lipocalins Rbp, Bbp or ApoD,which presently have already been exhaustively studied biochemically,are used. The use of lipocalins of human origin is especially preferredfor the production of anticalins. This especially applies when anapplication of the resulting anticalin(s) is intended for humans since,for example, in diagnostic or therapeutic applications in vivo, aminimal immunogenic effect is to be expected as compared to lipocalinsfrom other organisms. However, other lipocalins as well as lipocalinswhich, possibly, have yet to be discovered can prove to be especiallyadvantageous for the production of anticalins. Artificial proteins witha folding element which is structurally equivalent to the β-barrel ofthe lipocalins can also be used.

Preferably the anticalin molecules of the invention should be able tobind the desired ligand (e.g., human serum albumin) with a determinableaffinity, i.e., with an affinity constant of at least 10⁵ M⁻¹.Affinities lower than this are generally no longer exactly measurablewith common methods and are therefore of secondary importance forpractical applications. Especially preferred are anticalins which bindthe desired ligand with an affinity of at least 10⁶ M⁻¹, correspondingto a dissociation constant for the complex of 1 μM. The binding affinityof an anticalin to the desired ligand can be measured by the personskilled in the art by a multitude of methods, for example byfluorescence titration, by competition ELISA or by the technique ofsurface plasmon resonance.

The lipocalin cDNA, which can be produced and cloned by the personskilled in the art by known methods, can serve as a starting point formutagenesis of the peptide loop, as it was for example described for Bbp(Schmidt and Skerra, Eur. J. Biochem. 219 (1994), 855-863).Alternatively, genomic DNA can also be employed for gene synthesis or acombination of these methods can be performed. For the mutagenesis ofthe amino acids in the four peptide loops, the person skilled in the arthas at his disposal the various known methods for site-directedmutagenesis or for mutagenesis by means of the polymerase chainreaction. The mutagenesis method can, for example, be characterized inthat mixtures of synthetic oligodeoxynucleotides, which bear adegenerate base composition at the desired positions, can be used forintroduction of the mutations. The implementation of nucleotide buildingblocks with reduced base pair specificity, as for example inosine, isalso an option for the introduction of mutations into the chosensequence segment or amino acid positions. The procedure for mutagenesisof ligand-binding sites is simplified as compared to antibodies, sincefor the lipocalins only four instead of six sequencesegments—corresponding to the four above cited peptide loops—have to bemanipulated for this purpose.

In the methods of site-directed random mutagenesis implementingsynthetic oligodeoxynucleotides, the relevant amino acid positions inthe lipocalin structure which are to be mutated can be determined inadvance. The ideal selection of the amino acid positions to be mutatedcan depend on the one hand on the lipocalin used, and on the other handon the desired ligand (e.g., human serum albumin). It can be useful tomaintain the total number of mutated amino acid positions within asingle experiment low enough such that the collection of variantsobtained by mutagenesis, i.e. the so-called library, can in its totalityor, at least in a representative selection therefrom, be realized ascompletely as possible in its combinatorial complexity, not only at thelevel of the coding nucleic acids, but also at the level of the geneproducts.

It is possible to choose the amino acid positions to be mutated in ameaningful way especially when structural information exists pertainingto the lipocalin itself which is to be used, as is the case with BBP andRbp or at least pertaining to a lipocalin with a similar structure, asfor example in the case of ApoD. The set of amino acid positions chosencan further depend on the characteristics of the desired ligand. It canalso prove advantageous to exclude single amino acid positions in theregion of the ligand-binding pocket from mutagenesis if these, forexample, prove to be essential for the folding efficiency or the foldingstability of the protein. Specific oligonucleotide-based methods oflipocalin mutagenesis are described, for example, in U.S. PublicationNo. 20060058510, the entire contents of which are incorporated herein byreference.

After expressing the coding nucleic acid sequences subjected tomutagenesis, clones carrying the genetic information for anticalinswhich bind a given ligand (e.g., human serum albumin) can be selectedfrom the differing clones of the library obtained. Known expressionstrategies and selection strategies can be implemented for the selectionof these clones. Methods of this sort have been described in the contextof the production or the engineering of recombinant antibody fragments,such as the “phage display” technique or “colony screening” methods(Skerra et al., Anal. Biochem. 196 (1991), 151-155).

Descriptions of “phage display” techniques are found, for example, inHoess, Curr. Opin. Struct. Biol. 3 (1993), 572-579; Wells and Lowman,Curr. Opin. Struct. Biol. 2 (1992), 597-604; and Kay et al., PhageDisplay of Peptides and Proteins—A Laboratory Manual (1996), AcademicPress. Briefly, in an exemplary embodiment, phasmids are produced whicheffect the expression of the mutated lipocalin structural gene as afusion protein with a signal sequence at the N-terminus, preferably theOmpA-signal sequence, and with the coat protein pIII of the phage M13(Model and Russel, in “The Bacteriophages”, Vol. 2 (1988), Plenum Press,New York, 375-456) or fragments of this coat protein, which areincorporated into the phage coat, at the C-terminus. The C-terminalfragment ApIII of the phage coat protein, which contains only aminoacids 217 to 406 of the natural coat protein pIII, is preferably used toproduce the fusion proteins. Especially preferred is a C-terminalfragment from pIII in which the cysteine residue at position 201 ismissing or is replaced by another amino acid. Further description ofphage display methods, selection methods, etc., that can be applied tolipocalins in production of “anticalins” possessing specific bindingproperties is detailed in, for example, U.S. Publication No.20060058510, the entire contents of which are incorporated herein byreference.

Anticalins can be identified and produced, for example, using theabove-described methods, to possess high affinity for a given ligand(e.g., human serum albumin). Ligand binding constants of more than 10⁶M⁻¹ can be achieved for anticalins, even in cases where a novel ligandbears no structural relationship whatsoever to biliverdin IXy, theoriginal ligand of Bbp (refer to U.S. Publication No. 20060058510). Suchaffinities for novel ligands attainable with the anticalins arecomparable with the affinities which are known for antibodies from thesecondary immune response. Furthermore, there additionally exists thepossibility to subject the anticalins produced to a further, optionallypartial random mutagenesis in order to select variants of even higheraffinity from the new library thus obtained. Corresponding procedureshave already been described for the case of recombinant antibodyfragments for the purpose of an “affinity maturation” (Low et al., J.Mol. Biol. 260 (1996), 359-368; Barbas and Burton, Trends Biotechnol. 14(1996), 230-234) and can also be applied to anticalins in acorresponding manner by the person skilled in the art.

Staphylococcal Protein A (SPA)/Affibody Example 20: Generation ofDual-Specific Ligand Comprising a Serum Albumin-Binding Affibody(Staphylococcal Protein A (SPA)) Non-Immunoglobulin Scaffold ViaSelection of Serum Albumin Binding Moieties

The Z domain of staphylococcal protein A (SPA) is subjected to libraryselection and, optionally, affinity maturation techniques in order toproduce human serum albumin-binding SPA-derived non-immunoglobulinscaffold molecules (termed “affibodies”) for use in dual-specificligands of the invention.

Real-time binding analysis by Biacore is performed to assess whetherhuman serum albumin specifically binds to immobilized SPA polypeptide.(One of skill in the art will recognize that binding affinity can beassessed using any appropriate method, including, e.g., precipitation oflabeled human serum albumin, competitive Biacore assay, etc.) Followingdetection of no or low binding affinity (e.g., Kd values in the μM rangeor higher) of an unaltered SPA polypeptide for human serum albumin, atleast one of a number of strategies are employed to impart human serumalbumin binding properties to the SPA polypeptide, including one or moreof the following methods designed to impart and/or enhance bindingaffinity of the molecule for target antigen.

Human serum albumin binding of SPA scaffold polypeptide(s) is achievedand optimized via mutagenic methods, optionally in combination withparallel and/or iterative selection methods as described below and/or asotherwise known in the art. SPA scaffold polypeptide domains aresubjected to randomized and/or NNK mutagenesis, performed as describedinfra. Such mutagenesis is performed upon the entirety of the Z domainof the SPA polypeptide or upon specific sequences within the SPApolypeptide, e.g., upon 13 solvent-accessible surface residues of domainZ as identified in Nord et al. (1997 Nat. Biotechnol. 15: 772-77), andis optionally randomized in order to evolve new or improved human serumalbumin-binding polypeptides. PCR is optionally used to perform suchmethods of mutagenesis, resulting in the generation of sequencediversity across targeted sequences within the SPA polypeptides. (Suchapproaches are similar to those described infra for dAb librarygeneration.) In addition to random methods of mutagenesis, directedmutagenesis of targeted amino acid residues is employed where structuralinformation establishes specific amino acid residues of SPA polypeptidesto be critical to binding of human serum albumin. In certainembodiments, repertoires of mutant Z domain genes are assembled andinserted into a phagemid vector adapted for monovalent phage display.Libraries comprising, e.g., millions of transformants, are constructedusing, e.g., NN(G/T) or alternative (C/A/G)NN degeneracy formutagenesis.

SPA polypeptides engineered as described above are subjected to paralleland/or iterative selection methods to identify those SPA polypeptidesthat are optimized for human serum albumin binding. For example,following production of a library of mutagenized SPA polypeptidesequences, said library of polypeptides is displayed on phage andsubjected to multiple rounds of selection requiring serum albuminbinding and/or proliferation, as is described infra for selection ofserum albumin-binding dAbs from libraries of dAbs. Biopanning againstthe human serum albumin target protein is performed to achievesignificant enrichment for serum albumin binding SPA molecules. Selectedclones are subsequently expressed in E. coli and analyzed by SDS-PAGE,circular dichroism spectroscopy, and binding studies to human serumalbumin by biospecific interaction analysis. The SPA molecules(affibodies) that bind to human serum albumin are anticipated to have asecondary structure similar to the native Z domain and have micromolardissociation constants (Kd) for their respective targets in the range ofμM or better (e.g., nM or pM).

Optionally, selection is performed against serum albumin immobilized onimmunotubes or against biotinlyated serum albumin in solution.Optionally, binding affinity is determined using surface plasmonresonance (SPR) and the Biacore (Karlsson et al., 1991), using a Biacoresystem (Uppsala, Sweden), with fully optimized SPA-derived polypeptidesideally achieving human serum albumin binding affinity Kd values in thenM range or better.

Following identification of SPA polypeptides that bind human serumalbumin, such polypeptides are then used to generate dual-specificligand compositions by any of the methods described infra.

Staphylococcal Protein A (SPA) Affibody Polypeptides

Solvent-exposed surfaces of bacterial receptors can be targeted forrandom mutagenesis followed by phenotypic selection for purpose ofimparting, e.g., binding affinity for serum albumin to such receptormolecules. Such proteins can be unusually stable, which makes themsuitable for various applications (Alexander et al. (1992) Biochemistry31: 3597-3603). In particular, for bacterial receptors containing helixbundle structures, the conformation can be expected to be tolerant tochanges in the side chains of residues not involved in helix packinginterfaces. Examples of such molecules are the relatively small (58residues) IgG-binding domain B of staphylococcal protein A (SPA) and thesynthetic analogue of domain B, designated domain Z (Nilsson et al.(1987) Protein Engineering 1: 107-113).

The SPA-derived domain Z is the primary domain of SPA utilized as ascaffold for purpose of constructing domain variants with novel bindingproperties (refer to, e.g., WO 00/63243 and WO 95/19374, incorporatedherein by reference in their entireties). The SPA Z domain is a 58 aminoacid residue cysteine-free three-helix bundle domain that is used as ascaffold for construction of combinatorial phagemid libraries from whichvariants are selected that target desired molecules (e.g., human serumalbumin) using phage display technology (Nilsson et al. 1987 ProteinEng. 1: 107-113; Nord et al. 1997 Nat. Biotechnol. 15: 772-777; Nord etal. 2000 J. Biotechnol. 80: 45-54; Hansson et al. 1999 Immunotechnology4: 237-252; Eklund et al., 2002 Proteins 48: 454-462; Rönnmark et al.2002 Eur. J. Biochem. 269: 2647-2655). Such target-binding variants,termed “affibody” molecules, are selected as binders to target proteinsby phage display of combinatorial libraries in which typically 13side-chains on the surface of helices 1 and 2 (Q9, Q10, N11, F13, Y14,L17, H18, E24, E25, R27, N28, Q32 and K35) in the Z domain have beenrandomized (Lendel et al. 2006 J. Mol. Biol. 359: 1293-304). The simple,robust structure of such affibody molecules, together with their lowmolecular weight (7 Kda), make them suitable for a wide variety ofapplications. Documented efficacy has been shown in bioprocess- andlaboratory-scale bioseparations (Nord et al. 2000 J. Biotechnol. 80:45-54; Nord et al. 2001 Eur. J. Biochem. 268: 4269-4277; Gräslund et al.2002 J. Biotechnol. 99: 41-50), and promising results have been obtainedwhen evaluating affibody ligands as detection reagents (Karlström andNygren 2001 Anal. Biochem. 295: 22-30; Rönnmark et al. 2002 J. Immunol.Methods 261: 199-211), to engineer adenoviral tropism (Henning et al.2002 Hum. Gene Ther. 13: 1427-1439) and to inhibit receptor interactions(Sandstrom et al. 2003 Protein Eng. 16: 691-697). Thus, engineeredaffibody ligands that, e.g., bind to human serum albumin are desirablecomponents of certain dual-specific ligand compositions of the presentinvention.

Libraries of polypeptides derived from the Z domain of staphylococcalprotein A may be generated by any method of mutagenesis as known in theart and/or as described infra. Following creation of such polypeptidelibraries, variants capable of binding desired target molecules (e.g.,human serum albumin) can be efficiently selected and identified using,for example, in vitro selection technologies such as phage display (Dunn1996; Smith and Patrenko 1997; Hoogenboom et al. 1998), ribosomaldisplay (Hanes and Pluckthun 1997; He and Taussig 1997) peptides onplasmids (Schatz 1993) or bacterial display (Georgiou et al. 1997). Forsuch selections, a correlation between library size (complexity) and thelikelihood of isolating binders of higher affinities (KD=10⁻⁸ M orlower) has been theoretically considered (Perelson and Oster 1979) andexperimentally demonstrated (Griffiths et al. 1994; Vaughan et al. 1996;Aujame et al. 1997).

Affibodies have several advantages over traditional antibodies, e.g. (i)a lower cost of manufacture; (ii) smaller size; (iii) increasedstability and robustness; and (iv) the ability of being producedrecombinantly in a bacterial host, or by chemical synthesis, whichobviates the risk for viral contamination.

An affibody is a polypeptide which is a derivative of a staphylococcalprotein A (SPA) domain, said SPA domain being the B or Z domain, whereina number of the amino acid residues have been substituted by other aminoacid residues, said substitution being made without substantial loss ofthe basic structure and stability of the said SPA domain, and saidsubstitution resulting in interaction capacity of the said polypeptidewith at least one domain of a target antigen (e.g., human serumalbumin). The number of substituted amino acid residues could be from 1to about 30, or from 1 to about 13. Other possible ranges are from 4 toabout 30; from 4 to about 13; from 5 to about 20, or from 5 to about 13amino acid residues. It will be understood by the skilled person, e.g.,from Nord et al. 1997 Nat. Biotechnol. 15: 772-777, that preferentiallyamino residues located on the surface of the Z-domain can besubstituted, while the core of the bundle should be kept constant toconserve the structural properties of the molecule.

A process for the manufacture of an affibody is set forth, e.g., in WO00/63243, and for purposes of the present invention could involve, e.g.,the following steps: (i) displaying, by e.g. phage display (for areview, see, e.g., Kay, K. et al. (eds.) Phage Display of Peptides andProteins: A Laboratory Manual, Academic Press, San Diego, ISBN 0-12-402380-0), ribosomal display (for a review, see e.g. Hanes, J. et al. (1998)Proc. Natl. Acad. Sci. USA 95: 14130-14135) or cell display (for areview, see e.g. Daugherty, P. S. et al. (1998) Protein Eng. 11:825-832), polypeptide variants from a protein library embodying arepertoire of polypeptide variants derived from SPA domain B or Z; (ii)selecting clones expressing polypeptides that bind to human serumalbumin; and (iii) producing polypeptides by recombinant expression ofthe selected clones or by chemical synthesis.

Avimer Example 21: Generation of Dual-Specific Ligand Comprising a SerumAlbumin-Binding Avimer Via CDR Grafting

The CDR domains of dAb7h14 are used to construct an avimer polypeptidethat binds human serum albumin in the following manner. The CDR1(RASQWIGSQLS; SEQ ID NO.:95), CDR2 (WRSSLQS; SEQ ID NO.:96), and CDR3(AQGAALPRT; SEQ ID NO.:97) sequences of dAb7h14 are grafted into a C2monomer (described in US Patent Publication No. 2005/0221384,incorporated herein by reference in its entirety) at residues 17-28,49-53 and 78-85, respectively, which constitute the loop regions 1, 2and 3, respectively of the C2 monomer. Real-time binding analysis byBiacore is performed to assess whether human serum albumin specificallybinds to immobilized C2-derived monomer polypeptide comprising theanti-human serum albumin CDR domains of dAb7h14. (One of skill in theart will recognize that binding affinity can be assessed using anyappropriate method, including, e.g., precipitation of labeled humanserum albumin, competitive Biacore assay, etc.) If no or low human serumalbumin affinity (e.g., Kd values in the μM range or higher) isdetected, at least one of a number of strategies are employed to improvethe human serum albumin binding properties of the CDR-grafted C2 monomer(and/or of avimer dimers, trimers and other higher-order iterationcompositions), including any of the following methods that contribute tobinding affinity.

The length(s) of dAb7h14 CDR-grafted regions of the initial C2 monomerpolypeptide (and/or of iteratively-produced avimer dimer, trimer, etc.polypeptides) corresponding to solvent-exposed loop regions within thenative C2 monomer (and/or within other native monomers used in theavimer compositions) are adjusted through the use of linkerpolypeptides. For example, the nine amino acid residue CDR3 peptidesequence of dAb7h14 can be extended to 13 amino acid residues in lengthusing amino acid linkers of, e.g., zero to four residues in lengthlocated on either and/or both the N- or C-terminal flanks of the dAb7h14CDR3 polypeptide sequence, thereby achieving a total grafted peptidesequence length of 13 amino acids within the CDR3-grafted domaincorresponding to loop 3 of the C2 monomer polypeptide. Such use oflinker polypeptide(s) is optionally combined with mutagenesis of thelinker sequences, CDR sequences and/or non-CDR C2 monomer polypeptidesequences (e.g., using mutagenic optimization procedures as describedbelow), in order to improve the human serum albumin binding capabilityof CDR-grafted C2 monomer polypeptide(s) (e.g., via optimization of bothCDR and C2 monomer polypeptide sequences within the CDR-grafted C2monomer polypeptides). The polypeptide linkers employed for such purposeeither possess a predetermined sequence, or, optionally, are selectedfrom a population of randomized polypeptide linker sequences viaassessment of the human serum albumin binding capabilities oflinker-containing CDR-grafted C2 monomer polypeptides. Optimizationmethods are performed in parallel and/or iteratively. Both parallel anditerative optimization (e.g., affinity maturation) processes employselection methods as described below and/or as known in the art asuseful for optimization of polypeptide binding properties.

Human serum albumin binding of CDR-grafted C2 monomer polypeptide(s)(and/or of avimer dimer, trimer, etc. iteratively-produced higher-ordercompositions, or individual additional monomers contributing to same)presenting dAb7h14 CDRs is optimized via mutagenesis, optionally incombination with parallel and/or iterative selection methods asdescribed below and/or as otherwise known in the art. For the exemplaryC2 monomer scaffold polypeptide, domains surrounding grafted dAb7h14 CDRpolypeptide sequences are subjected to randomized and/or NNKmutagenesis, performed as described infra. Such mutagenesis isoptionally performed within the C2 monomer polypeptide sequence uponselected amino acid residues as set forth, e.g., in US PatentPublication No. 2005/0221384, or is optionally performed upon allnon-CDR amino acid residues, and is optionally randomized in order toevolve new or improved human serum albumin-binding polypeptides.Optionally, dAb7h14 CDR polypeptide domains presented within theCDR-grafted C2 monomer polypeptide are subjected to mutagenesis via,e.g., random mutagenesis, NNK mutagenesis, look-through mutagenesisand/or other art-recognized method. PCR is optionally used to performsuch methods of mutagenesis, resulting in the generation of sequencediversity across targeted sequences within the CDR-grafted C2 monomerpolypeptides. Such approaches are similar to those described infra fordAb library generation. In addition to random and/or look-throughmethods of mutagenesis, directed mutagenesis of targeted amino acidresidues is employed where structural information establishes specificamino acid residues to be critical to binding of human serum albumin.

C2 monomer polypeptides (and/or iteratively produced avimer compositionscomprising individual monomers) comprising grafted dAb7h14 CDR sequencesengineered as described above are subjected to parallel and/or iterativeselection methods to identify those C2 monomer polypeptides (and avimercompositions) that are optimized for human serum albumin binding. Forexample, following production of a library of dAb7h14 CDR-grafted C2monomer polypeptide sequences, this library of such polypeptides isdisplayed on phage and subjected to multiple rounds of selectionrequiring serum albumin binding and/or proliferation, as is describedinfra for selection of serum albumin-binding dAbs from libraries ofdAbs. Optionally, selection is performed against serum albuminimmobilized on immunotubes or against biotinlyated serum albumin insolution. Optionally, binding affinity is determined using surfaceplasmon resonance (SPR) and the Biacore (Karlsson et al., 1991), using aBiacore system (Uppsala, Sweden), with fully optimized avimerscomprising C2-derived monomers ideally achieving human serum albuminbinding affinity Kd values in the nM range or better.

Upon identification of C2 monomer-derived polypeptides that bind humanserum albumin, human serum binding properties of such initial monomersmay be further enhanced via combination of such monomers with othermonomers, followed by further mutagenesis and/or selection, therebyforming an avimer composition possessing specific affinity for humanserum albumin. Following identification of an avimer compositionpossessing affinity for human serum albumin, such avimer polypeptidesare then used to generate dual-specific ligand compositions by any ofthe methods described infra.

Example 22: Generation of Dual-Specific Ligand Comprising a SerumAlbumin-Binding Avimer Non-Immunoglobulin Scaffold Via Selection ofSerum Albumin Binding Moieties

The native C2 monomer polypeptide as set forth in is subjected tolibrary selection and, optionally, affinity maturation techniques, thencombined with an additional monomer (e.g., a fibronectin monomer, forwhich human serum albumin affinity optionally can be optimized inparallel) and optionally iteratively subjected to library selection and,optionally, affinity maturation techniques in order to produce a humanserum albumin-binding avimer non-immunoglobulin scaffold molecule foruse in dual-specific ligands of the invention.

Real-time binding analysis by Biacore is performed to assess whetherhuman serum albumin specifically binds to an immobilized C2 monomerpolypeptide (and/or an iteratively-produced avimer molecule). Followingdetection of no or low binding affinity (e.g., Kd values in the μM rangeor higher) of a C2 monomer polypeptide for human serum albumin, at leastone of a number of strategies are employed to impart human serum albuminbinding properties to the C2 monomer polypeptide, including one or moreof the following methods that contribute to binding affinity.

Human serum albumin binding of C2 monomer polypeptide(s) (and/oriteratively produced avimer dimer, trimer, etc. molecules) is achievedand optimized via mutagenic methods, optionally in combination withparallel and/or iterative selection methods as described below and/or asotherwise known in the art. C2 monomer polypeptide domains are subjectedto randomized and/or NNK mutagenesis, performed as described infra. Suchmutagenesis is performed upon the entirety of the C2 monomer polypeptideor upon specific sequences within the C2 monomer polypeptide uponselected amino acid residues as set forth, e.g., in US PatentPublication No. 2005/0221384, and is optionally randomized in order toevolve new or improved human serum albumin-binding polypeptides. PCR isoptionally used to perform such methods of mutagenesis, resulting in thegeneration of sequence diversity across targeted sequences within the C2monomer polypeptides and/or avimer molecules. (Such approaches aresimilar to those described infra for dAb library generation.) Inaddition to random methods of mutagenesis, directed mutagenesis oftargeted amino acid residues is employed where structural informationestablishes specific amino acid residues of C2 monomer and/or avimermolecules to be critical to binding of human serum albumin.

C2 monomer polypeptides engineered as described above are subjected toparallel and/or iterative selection methods to identify those C2 monomerpolypeptides and/or avimer molecules that are optimized for human serumalbumin binding. For example, following production of a library ofmutagenized C2 monomer polypeptide sequences, said library ofpolypeptides is displayed on phage and subjected to multiple rounds ofselection requiring serum albumin binding and/or proliferation, as isdescribed infra for selection of serum albumin-binding dAbs fromlibraries of dAbs. Optionally, the rounds of selection may includeiterations within which additional monomer subunits are added to form anew avimer molecule. Optionally, selection is performed against serumalbumin immobilized on immunotubes or against biotinlyated serum albuminin solution. Optionally, binding affinity is determined using surfaceplasmon resonance (SPR) and the Biacore (Karlsson et al., 1991), using aBiacore system (Uppsala, Sweden), with fully optimized avimerscomprising C2-derived monomer polypeptides ideally achieving human serumalbumin binding affinity Kd values in the nM range or better.

Upon identification of C2 monomer-derived polypeptides that bind humanserum albumin, human serum binding properties of such initial monomersmay be further enhanced via combination of such monomers with othermonomers, followed by further mutagenesis and/or selection, therebyforming an avimer composition possessing specific affinity for humanserum albumin. Following identification of an avimer compositionpossessing affinity for human serum albumin, such avimer polypeptidesare then used to generate dual-specific ligand compositions by any ofthe methods described infra.

Production and Use of Avimer Polypeptides

Avimers are evolved from a large family of human extracellular receptordomains by in vitro exon shuffling and phage display, generatingmultidomain proteins with binding and/or inhibitory properties. Linkingmultiple independent binding domains (selected, e.g., in iterativefashion for binding to a target protein, e.g., human serum albumin)creates avidity and results in improved affinity and specificitycompared with conventional single-epitope binding proteins. Otherpotential advantages include simple and efficient production ofmultitarget-specific molecules in E. coli, improved thermostability andresistance to proteases. Avimers can be produced that possess sub-nMaffinities against a target protein. For example, an avimer thatinhibits interleukin 6 with 0.8 pM IC₅₀ in cell-based assays has beenproduced and characterized as biologically active (Silverman et al. 2005Nature Biotechnology 23: 1556-1561; also see, for example, U.S. PatentApplication Publ. Nos. 2005/0221384, 2005/0164301, 2005/0053973 and2005/0089932, 2005/0048512, and 2004/0175756, each of which is herebyincorporated by reference herein in its entirety).

Avimer synthesis involves phage display libraries derived from the humanrepertoire of A domains. Synthetic recombination is used to create ahighly diverse pool of monomers, as described in Silverman et al. (2005Nature Biotechnology 23: 1556-1561). Following generation of a pool ofmonomers, the pool is screened against target protein (e.g., human serumalbumin). Initial candidates are identified, and an additional monomeris added and the resulting dimer library is screened against the targetprotein to identify candidate target-binding dimers. The method is theniterated to obtain a trimer with very high binding affinity for thetarget protein, and, optionally, may be iterated further to identifyhigher order candidate complexes. Candidate complexes that areidentified to bind with high affinity and specificity to target proteinsare termed avimers (for “avidity multimer”).

Monomer domains of avimers can be polypeptide chains of any size. Forexample, monomer domains can have about 25 to about 500, about 30 toabout 200, about 30 to about 100, about 90 to about 200, about 30 toabout 250, about 30 to about 60, about 9 to about 150, about 100 toabout 150, about 25 to about 50, or about 30 to about 150 amino acids.Similarly, a monomer domain of an avimer can comprise, e.g., from about30 to about 200 amino acids; from about 25 to about 180 amino acids;from about 40 to about 150 amino acids; from about 50 to about 130 aminoacids; or from about 75 to about 125 amino acids. Monomer domains andimmuno-domains can typically maintain stable conformation in solution.Sometimes, monomer domains of avimers and immuno-domains can foldindependently into a stable conformation. The stable conformation can bestabilized by metal ions. The stable conformation can optionally containdisulfide bonds (e.g., at least one, two, or three or more disulfidebonds). The disulfide bonds can optionally be formed between twocysteine residues.

Publications describing monomer domains and mosaic proteins andreferences cited within include the following: Hegyi, H and Bork, P.1997 J. Protein Chem., 16: 545-551; Baron et al. 1991 Trends Biochem.Sci. 16: 13-17; Ponting et al. 2000 Adv. Protein Chem. 54: 185-244;Doolittle 1995 Annu. Rev. Biochem 64: 287-314; Doolitte and Bork 1993Scientific American 269: 50-6; and Bork 1991 FEBS letters 286: 47-54.Monomer domains used in avimers can also include those domains found inPfam database and the SMART database. See Schultz et al. 2000 NucleicAcid Res. 28: 231-34.

Monomer domains that are particularly suitable for use in avimercompositions are (1) β-sandwich domains; (2) β-barrel domains; or (3)cysteine-rich domains comprising disulfide bonds. Cysteine-rich domainsemployed in avimers typically do not form an α-helix, a β-sheet, or aβ-barrel structure. Typically, the disulfide bonds promote folding ofthe domain into a three-dimensional structure. Usually, cysteine-richdomains have at least two disulfide bands, more typically at least threedisulfide bonds.

Monomer domains of avimers can have any number of characteristics. Forexample, the domains can have low or no immunogenicity in an animal(e.g., a human). Domains can have a small size, for example, the domainsmay be small enough to penetrate skin or other tissues. Domains canpossess a range of in vivo half-lives or stabilities.

Illustrative monomer domains suitable for use in avimer compositionsinclude, e.g., an EGF-like domain, a Kringle-domain, a fibronectin typeI domain, a fibronectin type II domain, a fibronectin type III domain, aPAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatictrypsin Inhibitor domain, a Kazal-type serine protease inhibitor domain,a Trefoil (P-type) domain, a von Willebrand factor type C domain, anAnaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat,LDL-receptor class A domain, a Sushi domain, a Link domain, aThrombospondin type I domain, a thyroglobulin monomer domain, anImmunoglobulin-like domain, a C-type lectin domain, a MAM domain, a vonWillebrand factor type A domain, a Somatomedin B domain, a WAP-type fourdisulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain, andother such domains known to those of ordinary skill in the art, as wellas derivatives and/or variants thereof. US Patent Publication No.20050221384 presents schematic diagrams of various exemplary forms ofmonomer domains found in molecules in the LDL-receptor family.

Suitable monomer domains (e.g., domains with the ability to foldindependently or with some limited assistance) can be selected from thefamilies of protein domains that contain β-sandwich or β-barrel threedimensional structures as defined by such computational sequenceanalysis tools as Simple Modular Architecture Research Tool (SMART; seeShultz et al. 2000 Nucleic Acids Research 28: 231-234) or CATH (seePearl et al. 2000 Nucleic Acids Research 28: 277-282). Exemplary monomerdomains of avimers also include domains of fibronectin type III domain,an anticalin domain and a Ig-like domain from CTLA-4. Some aspects ofthese domains are described in WO 01/64942 by Lipovsek et al.,WO99/16873 by Beste et al., and WO 00/60070 by Desmet et al., thecontents of which are incorporated in their entirety herein byreference.

Monomer domains of avimers are optionally cysteine rich. Suitablecysteine rich monomer domains include, e.g., the LDL receptor class Adomain (“A-domain”) or the EGF-like domain. The monomer domains can alsohave a cluster of negatively charged residues. Optionally, the monomerdomains contain a repeated sequence, such as YWTD as found in theβ-Propeller domain. Another exemplary monomer domain suitable for use inavimers is the C2 domain. Exemplary A domain and C2 domain sequences andconsensus sequences useful in avimer production, including exemplaryselections of amino acid residues (e.g., surface-exposed loop residues)most desirable for mutagenic targeting, are presented in US PatentPublication No. 2005/0221384.

Polynucleotides (also referred to as nucleic acids) encoding the monomerdomains are typically employed to make monomer domains via expression.Nucleic acids that encode monomer domains can be derived from a varietyof different sources. Libraries of monomer domains can be prepared byexpressing a plurality of different nucleic acids encoding naturallyoccurring monomer domains, altered monomer domains (i.e., monomer domainvariants), or a combinations thereof.

Monomer domains that bind to a selected or desired ligand (e.g., humanserum albumin) or mixture of ligands are identified, optionally as aninitial step in avimer production. In some embodiments, monomer domainsand/or immuno-domains are identified or selected for a desired property(e.g., binding affinity for human serum albumin) and then the monomerdomains and/or immuno-domains are formed into multimers. For thoseembodiments, any method resulting in selection of domains with a desiredproperty (e.g., human serum albumin binding) can be used. For example,the methods can comprise providing a plurality of different nucleicacids, each nucleic acid encoding a monomer domain; translating theplurality of different nucleic acids, thereby providing a plurality ofdifferent monomer domains; screening the plurality of different monomerdomains for binding of the desired ligand or a mixture of ligands; and,identifying members of the plurality of different monomer domains thatbind the desired ligand or mixture of ligands.

Monomer domains for avimer production can be naturally-occurring oraltered (non-natural variants). The term “naturally occurring” is usedherein to indicate that an object can be found in nature. For example,natural monomer domains can include human monomer domains or optionally,domains derived from different species or sources, e.g., mammals,primates, rodents, fish, birds, reptiles, plants, etc. The naturaloccurring monomer domains can be obtained by a number of methods, e.g.,by PCR amplification of genomic DNA or cDNA. The term “native”, as usedherein, is used in reference to a nucleic acid and/or polypeptide thathas not been altered via mutagenesis or otherwise via performance of anyof the methods described infra.

Monomer domains of avimers can be naturally-occurring domains ornon-naturally occurring variants. Libraries of monomer domains employedin synthesis of avimers may contain naturally-occurring monomer domain,non-naturally occurring monomer domain variants, or a combinationthereof.

A variety of reporting display vectors or systems can be used to expressnucleic acids encoding monomer domains and avimers, and to test for adesired activity (e.g., human serum albumin binding). For example, aphage display system is a system in which monomer domains are expressedas fusion proteins on the phage surface (Pharmacia, Milwaukee Wis.).Phage display can involve the presentation of a polypeptide sequenceencoding monomer domains and/or immuno-domains on the surface of afilamentous bacteriophage, typically as a fusion with a bacteriophagecoat protein. Exemplary methods of affinity enrichment and phage displayare set forth, for example, in PCT patent publication Nos. 91/17271,91/18980, and 91/19818 and 93/08278, incorporated herein by reference intheir entireties.

Examples of other display systems include ribosome displays, anucleotide-linked display (see, e.g., U.S. Pat. Nos. 6,281,344;6,194,550, 6,207,446, 6,214,553, and 6,258,558), cell surface displaysand the like. The cell surface displays include a variety of cells,e.g., E. coli, yeast and/or mammalian cells. When a cell is used as adisplay, the nucleic acids, e.g., obtained by PCR amplification followedby digestion, are introduced into the cell and translated. Optionally,polypeptides encoding monomer domains or avimers can be introduced,e.g., by injection, into the cell.

As described infra and in the art, avimers are multimeric compositions.In exemplary embodiments, multimers comprise at least two monomerdomains and/or immuno-domains. For example, multimers of the inventioncan comprise from 2 to about 10 monomer domains and/or immuno-domains,from 2 and about 8 monomer domains and/or immuno-domains, from about 3and about 10 monomer domains and/or immuno-domains, about 7 monomerdomains and/or immuno-domains, about 6 monomer domains and/orimmuno-domains, about 5 monomer domains and/or immuno-domains, or about4 monomer domains and/or immuno-domains. In some embodiments, themultimer comprises at least 3 monomer domains and/or immuno-domains.Typically, the monomer domains have been pre-selected for binding to thetarget molecule of interest (e.g., human serum albumin).

Within an avimer, each monomer domain may specifically bind to onetarget molecule (e.g., human serum albumin). Optionally, each monomerbinds to a different position (analogous to an epitope) on a targetmolecule. Multiple monomer domains and/or immuno-domains that bind tothe same target molecule can result in an avidity effect resulting inimproved avidity of the multimer avimer for the target molecule comparedto each individual monomer. Optionally, the multimer can possess anavidity of at least about 1.5, 2, 3, 4, 5, 10, 20, 50 or 100 times theavidity of a monomer domain alone for target protein (e.g., human serumalbumin).

Selected monomer domains can be joined by a linker to form a multimer(avimer). For example, a linker is positioned between each separatediscrete monomer domain in a multimer. Typically, immuno-domains arealso linked to each other or to monomer domains via a linker moiety.Linker moieties that can be readily employed to link immuno-domainvariants together are the same as those described for multimers ofmonomer domain variants. Exemplary linker moieties suitable for joiningimmuno-domain variants to other domains into multimers are describedherein.

Joining of selected monomer domains via a linker to form an avimer canbe accomplished using a variety of techniques known in the art. Forexample, combinatorial assembly of polynucleotides encoding selectedmonomer domains can be achieved by DNA ligation, or optionally, byPCR-based, self-priming overlap reactions. The linker can be attached toa monomer before the monomer is identified for its ability to bind to atarget multimer or after the monomer has been selected for the abilityto bind to a target multimer.

As mentioned above, the polypeptide(s) comprising avimers can bealtered. Descriptions of a variety of diversity generating proceduresfor generating modified or altered nucleic acid sequences encoding thesepolypeptides are described above and below in the following publicationsand the references cited therein: Soong, N. et al., Molecular breedingof viruses, (2000) Nat Genet 25(4):436-439; Stemmer, et al., Molecularbreeding of viruses for targeting and other clinical properties, (1999)Tumor Targeting 4:1-4; Ness et al., DNA Shuffling of subgenomicsequences of subtilisin, (1999) Nature Biotechnology 17:893-896; Changet al., Evolution of a cytokine using DNA family shuffling, (1999)Nature Biotechnology 17:793-797; Minshull and Stemmer, Protein evolutionby molecular breeding, (1999) Current Opinion in Chemical Biology3:284-290; Christians et al., Directed evolution of thymidine kinase forAZT phosphorylation using DNA family shuffling, (1999) NatureBiotechnology 17:259-264; Crameri et al., DNA shuffling of a family ofgenes from diverse species accelerates directed evolution, (1998) Nature391:288-291; Crameri et al., Molecular evolution of an arsenatedetoxification pathway by DNA shuffling, (1997) Nature Biotechnology15:436-438; Zhang et al., Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Patten et al., Applications of DNAShuffling to Pharmaceuticals and Vaccines, (1997) Current Opinion inBiotechnology 8:724-733; Crameri et al., Construction and evolution ofantibody-phage libraries by DNA shuffling, (1996) Nature Medicine2:100-103; Crameri et al., Improved green fluorescent protein bymolecular evolution using DNA shuffling, (1996) Nature Biotechnology14:315-319; Gates et al., Affinity selective isolation of ligands frompeptide libraries through display on a lac repressor ‘headpiece dimer’,(1996) Journal of Molecular Biology 255:373-386; Stemmer, Sexual PCR andAssembly PCR, (1996) In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer, Combinatorialmultiple cassette mutagenesis creates all the permutations of mutant andwildtype cassettes, (1995) BioTechniques 18:194-195; Stemmer et al.,Single-step assembly of a gene and entire plasmid form large numbers ofoligodeoxy-ribonucleotides, (1995) Gene, 164:49-53; Stemmer, TheEvolution of Molecular Computation, (1995) Science 270:1510; Stemmer.Searching Sequence Space, (1995) Bio/Technology 13:549-553; Stemmer,Rapid evolution of a protein in vitro by DNA shuffling, (1994) Nature370:389-391; and Stemmer, DNA shuffling by random fragmentation andreassembly: In vitro recombination for molecular evolution, (1994) Proc.Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al., Approaches to DNA mutagenesis:an overview, (1997) Anal Biochem. 254(2): 157-178; Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, (1996) Methods Mol. Biol. 57:369-374; Smith, In vitromutagenesis, (1985) Ann. Rev. Genet. 19:423-462; Botstein & Shortle,Strategies and applications of in vitro mutagenesis, (1985) Science229:1193-1201; Carter, Site-directed mutagenesis, (1986) Biochem. J.237:1-7; and Kunkel, The efficiency of oligonucleotide directedmutagenesis, (1987) in Nucleic Acids & Molecular Biology (Eckstein, F.and Lilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel, Rapid and efficient site-specificmutagenesis without phenotypic selection, (1985) Proc. Natl. Acad. Sci.USA 82:488-492; Kunkel et al., Rapid and efficient site-specificmutagenesis without phenotypic selection, (1987) Methods in Enzymol.154, 367-382; and Bass et al., Mutant Trp repressors with newDNA-binding specificities, (1988) Science 242:240-245);oligonucleotide-directed mutagenesis ((1983) Methods in Enzymol. 100:468-500; (1987) Methods in Enzymol. 154: 329-350; Zoller & Smith,Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment, (1982) Nucleic Acids Res. 10:6487-6500; Zoller &Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors, (1983) Methods in Enzymol. 100:468-500; and Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, (1987)Methods in Enzymol. 154:329-350); phosphorothioate-modified DNAmutagenesis (Taylor et al., The use of phosphorothioate-modified DNA inrestriction enzyme reactions to prepare nicked DNA, (1985) Nucl. AcidsRes. 13: 8749-8764; Taylor et al., The rapid generation ofoligonucleotide-directed mutations at high frequency usingphosphorothioate-modified DNA, (1985) Nucl. Acids Res. 13: 8765-8787;Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci Icleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, (1986) Nucl. Acids Res. 14:9679-9698; Sayers et al., Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis, (1988) Nucl. Acids Res.16:791-802; and Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al.,The gapped duplex DNA approach to oligonucleotide-directed mutationconstruction, (1984) Nucl. Acids Res. 12: 9441-9456; Kramer & FritzOligonucleotide-directed construction of mutations via gapped duplexDNA, (1987) Methods in Enzymol. 154:350-367; Kramer et al., Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations, (1988) Nucl. AcidsRes. 16: 7207; and Fritz et al., Oligonucleotide-directed constructionof mutations: a gapped duplex DNA procedure without enzymatic reactionsin vitro, (1988) Nucl. Acids Res. 16: 6987-6999).

Additional suitable methods include point mismatch repair (Kramer etal., Point Mismatch Repair, (1984) Cell 38:879-887), mutagenesis usingrepair-deficient host strains (Carter et al., Improved oligonucleotidesite-directed mutagenesis using M13 vectors, (1985) Nucl. Acids Res. 13:4431-4443; and Carter, Improved oligonucleotide-directed mutagenesisusing M13 vectors, (1987) Methods in Enzymol. 154: 382-403), deletionmutagenesis (Eghtedarzadeh & Henikoff, Use of oligonucleotides togenerate large deletions, (1986) Nucl. Acids Res. 14: 5115),restriction-selection and restriction-purification (Wells et al.,Importance of hydrogen-bond formation in stabilizing the transitionstate of subtilisin, (1986) Phil. Trans. R. Soc. Lond. A 317: 415-423),mutagenesis by total gene synthesis (Nambiar et al., Total synthesis andcloning of a gene coding for the ribonuclease S protein, (1984) Science223: 1299-1301; Sakamar and Khorana, Total synthesis and expression of agene for the a-subunit of bovine rod outer segment guaninenucleotide-binding protein (transducin), (1988) Nucl. Acids Res. 14:6361-6372; Wells et al., Cassette mutagenesis: an efficient method forgeneration of multiple mutations at defined sites, (1985) Gene34:315-323; and Grundstrom et al., Oligonucleotide-directed mutagenesisby microscale ‘shot-gun’ gene synthesis, (1985) Nucl. Acids Res. 13:3305-3316), double-strand break repair (Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, (1986) Proc.Natl. Acad. Sci. USA, 83:7177-7181; and Arnold, Protein engineering forunusual environments, (1993) Current Opinion in Biotechnology4:450-455). Additional details on many of the above methods can be foundin Methods in Enzymology Volume 154, which also describes usefulcontrols for trouble-shooting problems with various mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following U.S. patents, PCT publications and applications,and EPO publications: U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25,1997), “Methods for In Vitro Recombination;” U.S. Pat. No. 5,811,238 toStemmer et al. (Sep. 22, 1998) “Methods for Generating Polynucleotideshaving Desired Characteristics by Iterative Selection andRecombination;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3,1998), “DNA Mutagenesis by Random Fragmentation and Reassembly;” U.S.Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) “End-ComplementaryPolymerase Reaction;” U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov.17, 1998), “Methods and Compositions for Cellular and MetabolicEngineering;” WO 95/22625, Stemmer and Crameri, “Mutagenesis by RandomFragmentation and Reassembly;” WO 96/33207 by Stemmer and Lipschutz “EndComplementary Polymerase Chain Reaction;” WO 97/20078 by Stemmer andCrameri “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” WO 97/35966by Minshull and Stemmer, “Methods and Compositions for Cellular andMetabolic Engineering;” WO 99/41402 by Punnonen et al. “Targeting ofGenetic Vaccine Vectors;” WO 99/41383 by Punnonen et al. “AntigenLibrary Immunization;” WO 99/41369 by Punnonen et al. “Genetic VaccineVector Engineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination;” WO 00/18906 by Patten etal., “Shuffling of Codon-Altered Genes;” WO 00/04190 by del Cardayre etal. “Evolution of Whole Cells and Organisms by Recursive Recombination;”WO 00/42561 by Crameri et al., “Oligonucleotide Mediated Nucleic AcidRecombination;” WO 00/42559 by Selifonov and Stemmer “Methods ofPopulating Data Structures for Use in Evolutionary Simulations;” WO00/42560 by Selifonov et al., “Methods for Making Character Strings,Polynucleotides & Polypeptides Having Desired Characteristics;” WO01/23401 by Welch et al., “Use of Codon-Varied Oligonucleotide Synthesisfor Synthetic Shuffling;” and PCT/US01/06775 “Single-Stranded NucleicAcid Template-Mediated Recombination and Nucleic Acid FragmentIsolation” by Affholter.

The polypeptides (e.g., avimers) used in the present invention areoptionally expressed in cells. Multimer domains can be synthesized as asingle protein using expression systems well known in the art. Inaddition to the many texts noted above, general texts which describemolecular biological techniques useful herein, including the use ofvectors, promoters and many other topics relevant to expressing nucleicacids such as monomer domains, selected monomer domains, multimersand/or selected multimers, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—ALaboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 1999) (“Ausubel”)). Examples oftechniques sufficient to direct persons of skill through in vitroamplification methods, useful in identifying isolating and cloningmonomer domains and multimers coding nucleic acids, including thepolymerase chain reaction (PCR) the ligase chain reaction (LCR),Q-replicase amplification and other RNA polymerase mediated techniques(e.g., NASBA), are found in Berger, Sambrook, and Ausubel, as well asMullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis et al. eds) Academic Press Inc. SanDiego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826;Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990)Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringeret al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology13: 563-564. Improved methods of cloning in vitro amplified nucleicacids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods of amplifying large nucleic acids by PCR are summarized in Chenget al. (1994) Nature 369: 684-685 and the references therein, in whichPCR amplicons of up to 40 kb are generated. One of skill will appreciatethat essentially any RNA can be converted into a double stranded DNAsuitable for restriction digestion, PCR expansion and sequencing usingreverse transcriptase and a polymerase.

Vectors encoding, e.g., monomer domains and/or avimers may be introducedinto host cells, produced and/or selected by recombinant techniques.Host cells are genetically engineered (i.e., transduced, transformed ortransfected) with such vectors, which can be, for example, a cloningvector or an expression vector. The vector can be, for example, in theform of a plasmid, a viral particle, a phage, etc. The engineered hostcells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants, oramplifying the monomer domain, selected monomer domain, multimer and/orselected multimer gene(s) of interest. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to those skilled inthe art and in the references cited herein, including, e.g., Freshney(1994) Culture of Animal Cells, a Manual of Basic Technique, thirdedition, Wiley-Liss, New York and the references cited therein.

The polypeptides of the invention can also be produced in non-animalcells such as plants, yeast, fungi, bacteria and the like. Indeed, asnoted throughout, phage display is an especially relevant technique forproducing such polypeptides. In addition to Sambrook, Berger andAusubel, details regarding cell culture can be found in Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell,Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Avimers can also possess alterations of monomer domains, immuno-domainsand/or multimers that improve pharmacological properties, reduceimmunogenicity, or facilitate the transport of the multimer and/ormonomer domain into a cell or tissue (e.g., through the blood-brainbarrier, or through the skin). These types of alterations include avariety of modifications (e.g., the addition of sugar-groups orglycosylation), the addition of PEG, the addition of protein domainsthat bind a certain protein (e.g., HSA or other serum protein), theaddition of proteins fragments or sequences that signal movement ortransport into, out of and through a cell. Additional components canalso be added to a multimer and/or monomer domain to manipulate theproperties of the multimer and/or monomer domain. A variety ofcomponents can also be added including, e.g., a domain that binds aknown receptor (e.g., a Fc-region protein domain that binds a Fcreceptor), a toxin(s) or part of a toxin, a prodomain that can beoptionally cleaved off to activate the multimer or monomer domain, areporter molecule (e.g., green fluorescent protein), a component thatbind a reporter molecule (such as a radionuclide for radiotherapy,biotin or avidin) or a combination of modifications.

As used herein, “directed evolution” refers to a process by whichpolynucleotide variants are generated, expressed, and screened for anactivity (e.g., a polypeptide with binding activity for a human serumalbumin target protein) in a recursive process. One or more candidatesin the screen are selected and the process is then repeated usingpolynucleotides that encode the selected candidates to generate newvariants. Directed evolution involves at least two rounds of variationgeneration and can include 3, 4, 5, 10, 20 or more rounds of variationgeneration and selection. Variation can be generated by any method knownto those of skill in the art, including, e.g., by error-prone PCR, geneshuffling, chemical mutagenesis and the like.

The term “shuffling” is used herein to indicate recombination betweennon-identical sequences. In some embodiments, shuffling can includecrossover via homologous recombination or via non-homologousrecombination, such as via cre/lox and/or flp/frt systems. Shuffling canbe carried out by employing a variety of different formats, includingfor example, in vitro and in vivo shuffling formats, in silico shufflingformats, shuffling formats that utilize either double-stranded orsingle-stranded templates, primer based shuffling formats, nucleic acidfragmentation-based shuffling formats, and oligonucleotide-mediatedshuffling formats, all of which are based on recombination eventsbetween non-identical sequences and are described in more detail orreferenced herein below, as well as other similar recombination-basedformats.

The term “random” as used herein refers to a polynucleotide sequence oran amino acid sequence composed of two or more amino acids andconstructed by a stochastic or random process. The random polynucleotidesequence or amino acid sequence can include framework or scaffoldingmotifs, which can comprise invariant sequences.

GroEL and GroES Example 24: Generation of Dual-Specific LigandComprising a Serum Albumin-Binding Cpn10 (GroES) Non-ImmunoglobulinScaffold Via CDR Grafting

The CDR3 domain of dAb7h14 is used to construct a cpn10 (GroES)non-immunoglobulin scaffold polypeptide that binds human serum albuminin the following manner. The CDR3 (AQGAALPRT; SEQ ID NO.: ______)sequence of dAb7h14 is grafted into the cpn10 polypeptide in replacementof native cpn10 amino acid residues at positions 19-27 (mobile loopresidues). Real-time binding analysis by Biacore is performed to assesswhether human serum albumin specifically binds to immobilizedcpn10-derived polypeptide comprising the anti-human serum albumin CDR3domain of dAb7h14. (One of skill in the art will recognize that bindingaffinity can be assessed using any appropriate method, including, e.g.,precipitation of labeled human serum albumin, competitive Biacore assay,etc.) If no or low human serum albumin affinity (e.g., Kd values in theμM range or higher) is detected, at least one of a number of strategiesare employed to improve the human serum albumin binding properties ofthe CDR3-grafted cpn10 polypeptide, including any of the followingmethods that contribute binding affinity.

The length of the dAb7h14 CDR3-grafted region of the cpn10 polypeptidecorresponding to the mobile loop region within the native cpn10polypeptide is adjusted through deletion of amino acid residues and/orthe use of linker polypeptides. For example, the nine amino acid residueCDR3 peptide sequence of dAb7h14 is extended to 16 amino acid residuesin length using amino acid linkers of, e.g., zero to seven residues inlength located on either and/or both the N- or C-terminal flanks of thedAb7h14 CDR3 polypeptide sequence, thereby achieving a total graftedpeptide sequence length of 16 amino acids within the CDR3-grafted domaincorresponding to the mobile loop in the native cpn10 sequence. Such useof linker polypeptide(s) is optionally combined with mutagenesis of thelinker sequences, CDR3 sequence(s) and/or non-CDR cpn10 sequences (e.g.,using mutagenic optimization procedures as described below), in order toimprove the human serum albumin binding capability of CDR3-grafted cpn10polypeptides (e.g., via optimization of both CDR and fibronectinsequences within the CDR3-grafted cpn10 polypeptides). The polypeptidelinkers employed for such purpose either possess a predeterminedsequence, or, optionally, are selected from a population of randomizedpolypeptide linker sequences via assessment of the human serum albuminbinding capabilities of linker-containing CDR3-grafted cpn10polypeptides. Optimization methods are performed in parallel and/oriteratively. Both parallel and iterative optimization (e.g., affinitymaturation) processes employ selection methods as described below and/oras known in the art as useful for optimization of polypeptide bindingproperties.

Human serum albumin binding of CDR-grafted cpn10 polypeptide(s)presenting dAb7h14 CDR3 is optimized via mutagenesis, optionally incombination with parallel and/or iterative selection methods asdescribed below and/or as otherwise known in the art. Cpn10 scaffoldpolypeptide domains surrounding grafted dAb7h14 CDR3 polypeptidesequence are subjected to randomized and/or NNK mutagenesis, performedas described infra. Such mutagenesis is performed within the cpn10polypeptide sequence upon non-grafted amino acid residues, and isoptionally randomized in order to evolve new or improved human serumalbumin-binding polypeptides. Optionally, the dAb7h14 CDR3 polypeptidedomain presented within the CDR3-grafted cpn10 polypeptide is subjectedto mutagenesis via, e.g., random mutagenesis, NNK mutagenesis,look-through mutagenesis and/or other art-recognized method. PCR isoptionally used to perform such methods of mutagenesis, resulting in thegeneration of sequence diversity across targeted sequences within theCDR3-grafted cpn10 polypeptides. Such approaches are similar to thosedescribed infra for dAb library generation. In addition to random and/orlook-through methods of mutagenesis, directed mutagenesis of targetedamino acid residues is employed where structural information establishesspecific amino acid residues to be critical to binding of human serumalbumin.

Cpn10 polypeptides comprising grafted dAb7h14 CDR3 sequence engineeredas described above are subjected to parallel and/or iterative selectionmethods to identify those cpn10 polypeptides that are optimized forhuman serum albumin binding. For example, following production of alibrary of dAb7h14 CDR3-grafted cpn10 polypeptide sequences, thislibrary of such polypeptides is displayed on phage and subjected tomultiple rounds of selection requiring serum albumin binding and/orproliferation, as is described infra for selection of serumalbumin-binding dAbs from libraries of dAbs. Optionally, selection isperformed against serum albumin immobilized on immunotubes or againstbiotinlyated serum albumin in solution. Optionally, binding affinity isdetermined using surface plasmon resonance (SPR) and the Biacore(Karlsson et al., 1991), using a Biacore system (Uppsala, Sweden), withfully optimized monomeric and/or oligomeric cpn10-derived polypeptidesideally achieving human serum albumin binding affinity Kd values in thenM range or better.

Upon identification of monomeric cpn10-derived polypeptides that bindhuman serum albumin, human serum binding properties of such initialmonomers may be further enhanced via combination of such monomers withother monomers, followed by further mutagenesis and/or selection,thereby forming an oligomeric cpn10/GroES composition possessingspecific affinity for human serum albumin. Following identification ofan oligomeric cpn10/GroES composition possessing affinity for humanserum albumin, such polypeptides are then used to generate dual-specificligand compositions by any of the methods described infra.

Example 25: Generation of Dual-Specific Ligand Comprising a SerumAlbumin-Binding Cpn10 Non-Immunoglobulin Scaffold Via Selection of SerumAlbumin Binding Moieties

The native cpn10 polypeptide is subjected to library selection and,optionally, affinity maturation techniques in order to produce humanserum albumin-binding cpn10 non-immunoglobulin scaffold molecules foruse in dual-specific ligands of the invention.

The capability of a native cpn10 polypeptide to bind human serum albuminis initially ascertained via Biacore assay as described above. Followingdetection of no or low binding affinity (e.g., Kd values in the μM rangeor higher) of a cpn10 polypeptide for human serum albumin, at least oneof a number of strategies are employed to impart human serum albuminbinding properties to the cpn10 polypeptide, including one or more ofthe following methods that contribute to binding affinity.

Human serum albumin binding of cpn10 scaffold polypeptide(s) is achievedand optimized via mutagenic methods, optionally in combination withparallel and/or iterative selection methods as described below and/or asotherwise known in the art. Cpn10 scaffold polypeptide domains aresubjected to randomized and/or NNK mutagenesis, performed as describedinfra. Such mutagenesis is performed upon the entirety of the cpn10polypeptide or upon specific sequences within the cpn10 polypeptide,including mobile loop amino acid residues at positions 19-27, and isoptionally randomized in order to evolve new or improved human serumalbumin-binding polypeptides. PCR is optionally used to perform suchmethods of mutagenesis, resulting in the generation of sequencediversity across targeted sequences within the cpn10 polypeptides. (Suchapproaches are similar to those described infra for dAb librarygeneration.) In addition to random methods of mutagenesis, directedmutagenesis of targeted amino acid residues is employed where structuralinformation establishes specific amino acid residues of cpn10polypeptides to be critical to binding of human serum albumin.

Cpn10 polypeptides engineered as described above are subjected toparallel and/or iterative selection methods to identify those cpn10polypeptides that are optimized for human serum albumin binding. Forexample, following production of a library of mutagenized cpn10polypeptide sequences, said library of polypeptides is displayed onphage and subjected to multiple rounds of selection requiring serumalbumin binding and/or proliferation, as is described infra forselection of serum albumin-binding dAbs from libraries of dAbs.Optionally, selection is performed against serum albumin immobilized onimmunotubes or against biotinlyated serum albumin in solution.Optionally, binding affinity is determined using surface plasmonresonance (SPR) and the Biacore (Karlsson et al., 1991), using a Biacoresystem (Uppsala, Sweden), with fully optimized monomeric and/oroligomeric cpn10-derived polypeptides ideally achieving human serumalbumin binding affinity Kd values in the nM range or better.

Upon identification of monomeric cpn10-derived polypeptides that bindhuman serum albumin, human serum binding properties of such initialmonomers may be further enhanced via combination of such monomers withother monomers, followed by further mutagenesis and/or selection,thereby forming an oligomeric cpn10/GroES composition possessingspecific affinity for human serum albumin. Following identification ofan oligomeric cpn10/GroES composition possessing affinity for humanserum albumin, such polypeptides are then used to generate dual-specificligand compositions by any of the methods described infra.

GroEL Polypeptides

GroEL is a key molecular chaperone in E. coli that consists of 14subunits each of some 57.5 Kd molecular mass arranged in two sevenmembered rings (Braig et al. 1994 Proc. Natl. Acad. Sci. 90: 3978-3982).There is a large cavity in the GroEL ring system, and it is widelybelieved that the cavity is required for successful protein foldingactivity. For optimal activity, a co-chaperone, GroES, is required whichconsists of a seven membered ring of 10 Kd subunits (Hunt et al. 1996Nature 379: 37-45). Each GroES subunit uses a mobile loop with aconserved hydrophobic tripeptide for interaction with GroEL (Landry etal. 1993 Nature 364: 255-258). The mobile loops are generally less than16 amino acids in length and undergo a transition from disordered loopsto β-hairpins concomitant with binding the apical domains of GroEL(Shewmaker et al. 2001 J. Biol. Chem. 276: 31257-31264). The activity ofthe GroEL/GroES complex requires ATP. GroEL and GroES are widespreadthroughout all organisms, and often referred to as chaperonin (cpn)molecules, cpn60 and cpn10, respectively.

GroEL is an allosteric protein. Allosteric proteins are a special classof oligomeric proteins, which alternate between two or more differentthree-dimensional structures during binding of ligands and substrates.Allosteric proteins are often involved in control processes in biologyor where mechanical and physico-chemical energies are interconverted.The role of ATP is to trigger this allosteric change, causing GroEL toconvert from a state that binds denatured proteins tightly to one thatbinds denatured proteins weakly. The co-chaperone, GroES, aids in thisprocess by favoring the weak-binding state. It may also act as a cap,sealing off the cavity of GroEL. Further, its binding to GroEL is likelydirectly to compete with the binding of denatured substrates. The netresult is that the binding of GroES and ATP to GroEL which has asubstrate bound in its denatured form is to release the denaturedsubstrate either into the cavity or into solution where it can refold.

GroEL and GroES are polypeptide scaffolds that can be used tomultimerize monomeric polypeptides or protein domains, to producemultimeric proteins having any desired characteristic. As also describedinfra for, e.g., avimer compositions, it is often desirable tomultimerize polypeptide monomers.

Many proteins require the assistance of molecular chaperones in order tobe folded in vivo or to be refolded in vitro in high yields. Molecularchaperones are proteins, which are often large and require an energysource such as ATP to function. A key molecular chaperone in E. coli isGroEL, which consists of 14 subunits each of some 57. 5 Kda molecularmass arranged in two seven membered rings. There is a large cavity inthe GroEL ring system, and it is widely believed that the cavity isrequired for successful protein folding activity. For optimal activity,a co-chaperone, GroES, is required which consists of a seven memberedring of 10 Kda subunits. The activity of the GroEL/GroES complexrequires energy source ATP.

Minichaperones have been described in detail elsewhere (seeInternational patent application WO99/05163, the disclosure of which inincorporated herein by reference). Minichaperone polypeptides possesschaperoning activity when in monomeric form and do not require energy inthe form of ATP. Defined fragments of the apical domain of GroEL ofapproximately 143-186 amino acid residues in length have molecularchaperone activity towards proteins either in solution under monomericconditions or when monodispersed and attached to a support.

The GroEL and/or GroES scaffolds allow for the oligomerisation ofpolypeptides to form functional protein oligomers which have activitieswhich surpass those of recombinant monomeric polypeptides. Cpn10 is awidespread component of the cpn60/cpn10 chaperonin system. Examples ofcpn10 include bacterial GroES and bacteriophage T4 Gp31, and are alsolisted below. Further members of the cpn10 family will be known to thoseskilled in the art.

Protein scaffold subunits assemble to form a protein scaffold. Such ascaffold may have any shape and may comprise any number of subunits. Forcertain GroEL and GroES embodiments, the scaffold comprises between 2and 20 subunits, between 5 and 15 subunits, or about 10 subunits. Thenaturally-occurring scaffold structure of cpn10 family members comprisesseven subunits, in the shape of a seven-membered ring or annulus. Incertain embodiments, therefore, the scaffold is a seven-membered ring.

A heterologous amino acid sequence, which may be, e.g., a CDR3 domainderived from an antibody or antigen binding fragment thereof possessingaffinity for a target protein (e.g., human serum albumin) or,optionally, which may be a single residue such as cysteine which allowsfor the linkage of further groups or molecules to the scaffold, can beinserted into the sequence of the oligomerisable protein scaffoldsubunit such that both the N- and C-termini of the polypeptide monomerare formed by the sequence of the oligomerisable protein scaffoldsubunit. Thus, the heterologous polypeptide is included with thesequence of the scaffold subunit, for example by replacing one or moreamino acids thereof.

It is known that cpn10 subunits possess a “mobile loop” within theirstructure. The mobile loop is positioned between amino acids 15 and 34,preferably between amino acids 16 to 33, of the sequence of E. coliGroES, and equivalent positions on other members of the cpn10 family.The mobile loop of T4 Gp31 is located between residues 22 to 45,preferably 23 to 44. Optionally, the heterologous polypeptide can beinserted by replacing all or part of the mobile loop of a cpn10 familypolypeptide. Where the protein scaffold subunit is a cpn10 familypolypeptide, the heterologous sequence may moreover be incorporated atthe N- or C-terminus thereof, or in positions which are equivalent tothe roof b hairpin of cpn10 family peptides. This position is locatedbetween positions 54 and 67, preferably 55 to 66, and preferably 59 and61 of bacteriophage T4 Gp31, or between positions 43 to 63, preferably44 to 62, advantageously 50 to 53 of E. coli GroES.

Optionally, a polypeptide may be inserted at the N- or C-terminus of ascaffold subunit in association with circular permutation of the subunititself. Circular permutation is described in Graf and Schachman,PNAS(USA) 1996, 93: 11591. Essentially, the polypeptide is circularizedby fusion of the existing N- and C-termini, and cleavage of thepolypeptide chain elsewhere to create novel N- and C-termini. In apreferred embodiment of the invention, the heterologous polypeptide maybe included at the N- and/or C-terminus formed after circularpermutation. The site of formation of the novel termini may be selectedaccording to the features desired, and may include the mobile loopand/or the roof β hairpin.

Advantageously, heterologous sequences, which may be the same ordifferent, may be inserted at more than one of the positions and/or atdifferent positions than the above-identified positions within theprotein scaffold subunit. Thus, each subunit may comprise two or moreheterologous polypeptides, which are displayed on the scaffold when thisis assembled. Heterologous polypeptides may be displayed on a scaffoldsubunit in free or constrained form, depending on the degree of freedomprovided by the site of insertion into the scaffold sequence. Forexample, varying the length of the sequences flanking the mobile or βhairpin loops in the scaffold will modulate the degree of constraint ofany heterologous polypeptide inserted therein.

GroEL and/or GroES compositions also may comprise a polypeptide oligomercomprising two or more monomers. The oligomer may be configured as aheterooligomer, comprising two or more different amino acid sequencesinserted into the scaffold, or as a homooligomer, in which the sequencesinserted into the scaffold are the same.

The monomers which constitute the oligomer may be covalently crosslinkedto each other. Crosslinking may be performed by recombinant approaches,such that the monomers are expressed ab initio as an oligomer;alternatively, crosslinking may be performed at Cys residues in thescaffold. For example, unique Cys residues inserted between positions 50and 53 of the GroES scaffold, or equivalent positions on other membersof the cpn10 family, may be used to cross-link scaffold subunits.

The nature of the heterologous polypeptide inserted into the scaffoldsubunit may be selected at will. In certain embodiments, scaffoldproteins are synthesized which display antibodies or fragments thereofsuch as scFv, natural or camelised VH domains and VH CDR3 fragments.

In an exemplary embodiment, a polypeptide monomer capable ofoligomerisation can be prepared as described above and/or as set forthin WO 00/69907, incorporated herein by reference in its entirety. Themethod of such preparation can comprise insertion of a nucleic acidsequence encoding a heterologous polypeptide into a nucleic acidsequence encoding a subunit of an oligornerisable protein scaffold,incorporating the resulting nucleic acid into an expression vector, andexpressing the nucleic acid to produce the polypeptide monomers.Optionally, a polypeptide oligomer may then be produced via a processthat comprises allowing the polypeptide monomers produced as above toassociate into an oligomer. In certain embodiments, the monomers arecross-linked to form the oligomer.

In certain embodiments, a scaffold polypeptide is based on members ofthe cpn10/Hsp10 family, such as GroES or an analogue thereof. A highlypreferred analogue is the T4 polypeptide Gp31. GroES analogues,including Gp31, possess a mobile loop (Hunt, J. F., et al., (1997) Cell90, 361-371; Landry, S. J., et al., (1996) Proc. Natl. Acad. Sci. U.S.A.93, 11622-11627) which may be inserted into, or replaced, in order tofuse the heterologous polypeptide to the scaffold.

Cpn10 homologues are widespread throughout animals, plants and bacteria.For example, a search of GenBank indicates that cpn10 homologues areknown in the following species: Actinobacillus actinomycetemcomitans;Actinobacillus pleuropneumoniae; Aeromonas salmonicida; Agrobacteriumtumefaciens; Allochromatium vinosum; Amoeba proteus symbiotic bacterium;Aqui/ex aeolicus; Arabidopsis thaliana; Bacillus sp; Bacillusstearothermophilus; Bacillus subtilis; Bartonella henselae; Bordetellapertussis; Borrelia burgdorferi; Brucella abortus; Buchnera aphidicola;Burkholderia cepacia; Burkholderia vietnamiensis; Campylobacterjejuni;Caulobacter crescentus; Chlamydia muridarum; Chlamydia trachomatis;Chlamydophila pneumoniae; Clostridium acetobutylicum; Clostridiumperfringens; Clostridium thermocellum; coliphage T-Cowdria ruminantium;Cyanelle Cyanophora paradoxa; Ehrlichia canis; Ehrlichia chaffeensis;Ehrlichia equi; Ehrlichia phagocytophila; Ehrlichia risticii; Ehrlichiasennetsu; Ehrlichia sp ‘HGE agent; Enterobacter aerogenes; Enterobacteragglomerans; Enterobacter amnigenus; Enterobacter asburiae; Enterobactergergoviae; Enterobacter intermedius; Erwinia aphidicola; Erwiniacarotovora; Erwinia herbicola; Escherichia coli; Francisella tularensis;Glycine max; Haemophilus ducreyi; Haemophilus influenzae Rd,Helicobacter pylori; Holospora obtusa; Homo sapiens; Klebsiellaornithinolytica; Klebsiella oxytoca; Klebsiella planticola; Klebsiellapneumoniae; Lactobacillus helvetictis; LactobacillUS 7eae; Lactococcuslactis; Lawsonia intracellularis; Leptospira interrogans; Methylovorussp strain SS; Mycobacterium avium; Mycobacterium avium subsp avium;Mycobacterium avium subsp paratuberculosis; Mycobacterium leprae;Mycobacterium tuberculosis; Mycoplasma genitalium; Mycoplasmapneumoniae; Myzus persicae primary endosymbiont; Neisseria gonorrhoeae;Oscillatoria sp NKBG,—Pantoea ananas; Pasteurella multocida;Porphyromonas gingivalis; Pseudomonas aeruginosa; Pseudomonasaeruginosa; Pseudomonas putida; Rattus norvegicus; Rattus norvegicus;Rhizobium leguminosarum; Rhodobacter capsulatus; Rhodobactersphaeroides; Rhodothermus marinus; Rickettsia prowazekii; Rickettsiarickettsia; Saccharomyces cerevisiae; Serratia ficaria; Serratiamarcescens; Serratia rubidaea; Sinorhizobium meliloti; Sitophilus oryzaeprincipal endosymbiont; Stenotrophomonas maltophilia; Streptococcuspneumoniae; Streptomyces albus; Streptomyces coelicolor; Streptomycescoelicolor; Streptomyces lividans; Synechococcus sp; Synechococcusvulcanus; Synechocystis sp; Thermoanaerobacter brockii; Thermotogamaritima; Thermus aquaticus; Treponema pallidum; Wolbachia sp; Zymomonasmobilis.

An advantage of cpn10 family subunits is that they possess a mobileloop, responsible for the protein folding activity of the naturalchaperonin, which may be removed without affecting the scaffold. Cpn10with a deleted mobile loop possesses no biological activity, making itan advantageously inert scaffold, thus minimizing any potentiallydeleterious effects.

Insertion of an appropriate biologically active polypeptide can confer abiological activity (e.g., human serum albumin binding) on the novelpolypeptide thus generated. Indeed, the biological activity of theinserted polypeptide may be improved by incorporation of thebiologically active polypeptide into the scaffold, especially, e.g.,when mutagenesis and affinity-based screening methods as describedherein are used to optimize target protein binding of ascaffold-presented polypeptide.

Alternative sites for peptide insertion are possible. An advantageousoption is in the position equivalent to the roof β hairpin in GroES.This involves replacement of Glu- in Gp31 by the desired peptide. Theamino acid sequence is Pro (59)-Glu(60)-Gly(61). This is convenientlyconverted to a Smal site at the DNA level (CCC:GGG) encoding Pro-Gly,leaving a blunt-ended restriction site for peptide insertion as a DNAfragment. Similarly, an insertion may be made at between positions 50and 53 of the GroES sequence, and at equivalent positions in other cpn10family members. Alternatively, inverse PCR may be used, to display thepeptide on the opposite side of the scaffold.

Members of the cpn60/Hsp60 family of chaperonin molecules may also beused as scaffolds. For example, the tetradecameric bacterial chaperoninGroEL may be used. In certain embodiments, heterologous polypeptideswould be inserted between positions 191 and 376, in particular betweenpositions 197 and 333 (represented by SacII engineered and unique Cla Isites) to maintain intact the hinge region between the equatorial andthe apical domains in order to impart mobility to the insertedpolypeptide. The choice of scaffold may depend upon the intendedapplication of the oligomer (or dual-specific ligand comprising and/orderived from such an oligomer): for example, if the oligomer is intendedfor vaccination purposes, the use of an immunogenic scaffold, such asthat derived from Mycobacterium tuberculosis, is highly advantageous andconfers an adjuvant effect.

Mutants of cpn60 molecules can also be used. For example, the singlering mutant of GroEL (GroELSRI) contains four point mutations whicheffect the major attachment between the two rings of GroEL (R452E,E461A, S463A and V464A) and is functionally inactive in vitro because itis released to bind GroES. GroELSR2 has an additional mutation atGlu191-Gly, which restores activity by reducing the affinity for GroES.Both of these mutants form ring structures and would be suitable for useas scaffolds.

Certain naturally-occurring scaffold molecules are bacteriophageproducts: for this reason, naturally occurring antibodies to suchscaffolds are rare. This enhances the use of scaffold fusions as vaccineagents. T4 Gp31 with a deleted loop has no biological activity (exceptas a dominant-negative or intracellular vaccine against T4bacteriophage) thus minimizing deleterious effects on the host. However,insertion of appropriate sequences encoding polypeptides can conferbiological activity on the novel proteins. Indeed, the biologicalactivity may be improved by insertion into the scaffold protein.

The affinity of antibodies or antibody fragments for antigens (e.g.,human serum albumins) may be increased by oligomerisation according tothe present invention. Antibody fragments may be fragments such as Fv,Fab and F(ab′)₂ fragments or any derivatives thereof, such as a singlechain Fv fragments. The antibodies or antibody fragments may benon-recombinant, recombinant or humanized. The antibody may be of anyimmunoglobulin isotype, e.g., IgG, IgM, and so forth.

In certain embodiments, the antibody fragments may be camelised VHdomains. It is known that the main intermolecular interactions betweenantibodies and their cognate antigens are mediated through VH CDR3.

Use of GroEL and/or GroES (cpn10) scaffold molecules as described infraand as known in the art provides for the oligomerisation Of VH domains,or VH CDR3 domains, to produce a high-affinity oligomer. Two or moredomains may be included in such an oligomer; in an oligomer based on acpn10 scaffold, up to 7 domains may be included, forming a hetpamericoligomeric molecule (heptabody) that binds to a target protein (e.g.,human serum albumin).

For purpose of imparting and/or optimizing the affinity of certainscaffold polypeptides/oligomers for a target protein (e.g., human serumalbumin), variation may be introduced into heterologous polypeptidesinserted into scaffold polypeptides, such that the specificity and/oraffinity of such polypeptides/oligomers for their ligands/substrates canbe examined and/or mapped. Variants may be produced of the same loop, ora set of standard different loops may be devised, in order to assessrapidly the affinity of a novel polypeptide for target protein (e.g.,human serum albumin). Variants may be produced by randomization ofsequences according to known techniques, such as PCR. They may besubjected to selection by a screening protocol, such as phage display,before incorporation into protein scaffolds.

An “oligomerisable scaffold”, as referred to herein, is a polypeptidewhich is capable of oligomerising or being oligomerised to form ascaffold and to which a heterologous polypeptide may be fused,preferably covalently, without abolishing the oligomerisationcapabilities. Thus, it provides a “scaffold” using which polypeptidesmay be arranged into multimers in accordance with the present invention.Optionally, parts of the wild-type polypeptide from which the scaffoldis derived may be removed, for example by replacement with theheterologous polypeptide which is to be presented on the scaffold.

Monomers are polypeptides which possess the potential to oligomerise orto be oligomerised. Oligomerisation can be brought about by theincorporation, in the polypeptide, of an oligomerisable scaffold subunitwhich will oligomerise with further scaffold subunits if combinedtherewith. Optionally, oligomerisation can be brought about via use ofart-recognized linkers for purpose of joining together monomers.

As used herein, “oligomer” is synonymous with “polymer” or “multimer”and is used to indicate that the object in question is not monomeric.Thus, oligomeric polypeptides comprise at least two monomeric unitsjoined together covalently or non-covalently. The number of monomericunits employed will depend on the intended use of the oligomer, and maybe between 2 and 20 or more. Optionally, it is between 5 and 10, andpreferably about 7.

Phage Display

Phage display technology has proved to be enormously useful inbiological research. It enables ligands to be selected from largelibraries of molecules. Scaffold technology can harness the power ofphage display in a uniquely advantageous manner. Cpn10 molecules can bedisplayed as monomers on fd bacteriophages, similar to single-chain Fvmolecule display. Libraries of insertions (in place of the highly mobileloop, e.g., using CDR3 polypeptides derived from human serumalbumin-binding antibodies) are constructed by standard methods, and theresulting libraries screened for molecules of interest. Such selectionis affinity-based. After identification of molecules that possessaffinity for target protein (e.g., human serum albumin), potentially viaone or more iterations of mutagenesis, expression (the GroEL proteins,−57.5 Kda GroEL and −10 Kda GroES, can be expressed and purified aspreviously described (Chatellier et al. 1998 Proc. Natl. Acad. Sci. USA95: 9861-9866; Corrales and Fersht 1996 1: 265-273), or by anyart-recognized method) and affinity screening, such molecules can beoligomerised, thereby taking advantage of the avidity of such molecules.Optionally, certain selected monomers will be able to crosslink oroligomerise their binding partners.

Fibronectin Example 26: Generation of Dual-Specific Ligand Comprising aSerum Albumin-Binding Fibronectin Non-Immunoglobulin Scaffold Via CDRGrafting

The CDR domains of dAb7h14 are used to construct a fibronectinnon-immunoglobulin scaffold polypeptide that binds human serum albuminin the following manner. The CDR1 (RASQWIGSQLS; SEQ ID NO.:95), CDR2(WRSSLQS; SEQ ID NO.:96), and CDR3 (AQGAALPRT; SEQ ID NO.:97) sequencesof dAb7h14 are grafted into ¹⁰Fn3 in replacement of native ¹⁰Fn3 aminoacid residues at positions 21-31 (the BC loop), 51-56 (the DE loop), and76-88 (the FG loop), respectively. Real-time binding analysis by Biacoreis performed to assess whether human serum albumin specifically binds toimmobilized fibronectin-derived polypeptide comprising the anti-humanserum albumin CDR domains of dAb7h14. (One of skill in the art willrecognize that binding affinity can be assessed using any appropriatemethod, including, e.g., precipitation of labeled human serum albumin,competitive Biacore assay, etc.) If no or low human serum albuminaffinity (e.g., Kd values in the μM range or higher) is detected, atleast one of a number of strategies are employed to improve the humanserum albumin binding properties of the CDR-grafted fibronectinpolypeptide, including any of the following methods that contribute tobinding affinity.

The length(s) of dAb7h14 CDR-grafted regions of the fibronectinpolypeptide corresponding to solvent-exposed loop regions within thenative fibronectin polypeptide are adjusted through the use of linkerpolypeptides. For example, the nine amino acid residue CDR3 peptidesequence of dAb7h14 is extended to 13 amino acid residues in lengthusing amino acid linkers of, e.g., zero to four residues in lengthlocated on either and/or both the N- or C-terminal flanks of the dAb7h14CDR3 polypeptide sequence, thereby achieving a total grafted peptidesequence length of 13 amino acids within the CDR3-grafted domaincorresponding to the FG loop in the native fibronectin sequence. Suchuse of linker polypeptide(s) is optionally combined with mutagenesis ofthe linker sequences, CDR sequences and/or non-CDR fibronectin sequences(e.g., using mutagenic optimization procedures as described below), inorder to improve the human serum albumin binding capability ofCDR-grafted fibronectin polypeptides (e.g., via optimization of both CDRand fibronectin sequences within the CDR-grafted fibronectinpolypeptides). The polypeptide linkers employed for such purpose eitherpossess a predetermined sequence, or, optionally, are selected from apopulation of randomized polypeptide linker sequences via assessment ofthe human serum albumin binding capabilities of linker-containingCDR-grafted fibronectin polypeptides. Optimization methods are performedin parallel and/or iteratively. Both parallel and iterative optimization(e.g., affinity maturation) processes employ selection methods asdescribed below and/or as known in the art as useful for optimization ofpolypeptide binding properties.

Human serum albumin binding of CDR-grafted fibronectin polypeptide(s)presenting dAb7h14 CDRs is optimized via mutagenesis, optionally incombination with parallel and/or iterative selection methods asdescribed below and/or as otherwise known in the art. ¹⁰Fn3 scaffoldpolypeptide domains surrounding grafted dAb7h14 CDR polypeptidesequences are subjected to randomized and/or NNK mutagenesis, performedas described infra. Such mutagenesis is performed within the ¹⁰Fn3polypeptide sequence upon amino acids 1-9, 44-50, 61-54, 82-94 (edges ofbeta sheets); 19, 21, 30-46 (even), 79-65 (odd) (solvent-accessiblefaces of both beta sheets); and 14-16 and 36-45 (non-CDR-likesolvent-accessible loops and beta turns), and is optionally randomizedin order to evolve new or improved human serum albumin-bindingpolypeptides. Optionally, dAb7h14 CDR polypeptide domains presentedwithin the CDR-grafted fibronectin polypeptide are subjected tomutagenesis via, e.g., random mutagenesis, NNK mutagenesis, look-throughmutagenesis and/or other art-recognized method. PCR is optionally usedto perform such methods of mutagenesis, resulting in the generation ofsequence diversity across targeted sequences within the CDR-graftedfibronectin polypeptides. Such approaches are similar to those describedinfra for dAb library generation. In addition to random and/orlook-through methods of mutagenesis, directed mutagenesis of targetedamino acid residues is employed where structural information establishesspecific amino acid residues to be critical to binding of human serumalbumin.

Fibronectin polypeptides comprising grafted dAb7h14 CDR sequencesengineered as described above are subjected to parallel and/or iterativeselection methods to identify those fibronectin polypeptides that areoptimized for human serum albumin binding. For example, followingproduction of a library of dAb7h14 CDR-grafted fibronectin polypeptidesequences, this library of such polypeptides is displayed on phage andsubjected to multiple rounds of selection requiring serum albuminbinding and/or proliferation, as is described infra for selection ofserum albumin-binding dAbs from libraries of dAbs. Optionally, selectionis performed against serum albumin immobilized on immunotubes or againstbiotinlyated serum albumin in solution. Optionally, binding affinity isdetermined using surface plasmon resonance (SPR) and the Biacore(Karlsson et al., 1991), using a Biacore system (Uppsala, Sweden), withfully optimized fibronectin-derived polypeptides ideally achieving humanserum albumin binding affinity Kd values in the nM range or better.Following identification of fibronectin-derived polypeptides that bindhuman serum albumin, such polypeptides are then used to generatedual-specific ligand compositions by any of the methods described infra.

Example 27: Generation of Dual-Specific Ligand Comprising a SerumAlbumin-Binding Fibronectin Non-Immunoglobulin Scaffold Via Selection ofSerum Albumin Binding Moieties

The native fibronectin protein—specifically the ¹⁰Fn3 polypeptide offibronectin—is subjected to library selection and, optionally, affinitymaturation techniques in order to produce human serum albumin-bindingfibronectin non-immunoglobulin scaffold molecules for use indual-specific ligands of the invention.

Real-time binding analysis by Biacore is performed to assess whetherhuman serum albumin specifically binds to immobilized native fibronectinand/or fibronectin-derived polypeptide. Following detection of no or lowbinding affinity (e.g., Kd values in the • M range or higher) of afibronectin polypeptide for human serum albumin, at least one of anumber of strategies are employed to impart human serum albumin bindingproperties to the fibronectin polypeptide, including one or more of thefollowing methods that contribute to binding affinity.

Human serum albumin binding of fibronectin scaffold polypeptide(s) isachieved and optimized via mutagenic methods, optionally in combinationwith parallel and/or iterative selection methods as described belowand/or as otherwise known in the art. ¹⁰Fn3 scaffold polypeptide domainsare subjected to randomized and/or NNK mutagenesis, performed asdescribed infra. Such mutagenesis is performed upon the entirety of the¹⁰Fn3 polypeptide or upon specific sequences within the ¹⁰Fn3polypeptide, including amino acids 1-9, 44-50, 61-54, 82-94 (edges ofbeta sheets); 19, 21, 30-46 (even), 79-65 (odd) (solvent-accessiblefaces of both beta sheets); 21-31, 51-56, 76-88 (CDR-likesolvent-accessible loops); and 14-16 and 36-45 (other solvent-accessibleloops and beta turns), and is optionally randomized in order to evolvenew or improved human serum albumin-binding polypeptides. PCR isoptionally used to perform such methods of mutagenesis, resulting in thegeneration of sequence diversity across targeted sequences within thefibronectin polypeptides. (Such approaches are similar to thosedescribed infra for dAb library generation.) In addition to randommethods of mutagenesis, directed mutagenesis of targeted amino acidresidues is employed where structural information establishes specificamino acid residues of fibronectin polypeptides to be critical tobinding of human serum albumin.

Fibronectin polypeptides engineered as described above are subjected toparallel and/or iterative selection methods to identify thosefibronectin polypeptides that are optimized for human serum albuminbinding. For example, following production of a library of mutagenizedfibronectin polypeptide sequences, said library of polypeptides isdisplayed on phage and subjected to multiple rounds of selectionrequiring serum albumin binding and/or proliferation, as is describedinfra for selection of serum albumin-binding dAbs from libraries ofdAbs. Optionally, selection is performed against serum albuminimmobilized on immunotubes or against biotinlyated serum albumin insolution. Optionally, binding affinity is determined using surfaceplasmon resonance (SPR) and the Biacore (Karlsson et al., 1991), using aBiacore system (Uppsala, Sweden), with fully optimizedfibronectin-derived polypeptides ideally achieving human serum albuminbinding affinity Kd values in the nM range or better.

Following identification of fibronectin polypeptides that bind humanserum albumin, such polypeptides are then used to generate dual-specificligand compositions by any of the methods described infra.

Fibronectin Non-Immunoglobulin Scaffolds

In certain embodiments of the invention, a non-immunoglobulin scaffoldcomprising fibronectin, or a functional moiety and/or fragment thereof,is engineered to bind serum albumin. A non-immunoglobulin scaffoldstructure derived from the fibronectin type III module (Fn3) is used.The fibronectin type III module is a common domain found in mammalianblood and structural proteins, that occurs more than 400 times in theprotein sequence database and is estimated to occur in 2% of allproteins sequenced to date. Proteins that include an Fn3 module sequenceinclude fibronectins, tenascin, intracellular cytoskeletal proteins, andprokaryotic enzymes (Bork and Doolittle, Proc. Natl. Acad. Sci. USA89:8990, 1992; Bork et al., Nature Biotech. 15:553, 1997; Meinke et al.,J. Bacteriol. 175:1910, 1993; Watanabe et al., J. Biol. Chem. 265:15659,1990). A particular non-immunoglobulin scaffold of fibronectin is thetenth module of human Fn3 (¹⁰Fn3), which comprises 94 amino acidresidues. The overall fold of this domain is closely related to that ofthe smallest functional antibody fragment, the variable region of theheavy chain, which comprises the entire antigen recognition unit incamel and llama IgG. The major differences between camel and llamadomains and the ¹⁰Fn3 domain are that (i)¹⁰Fn3 has fewer beta strands(seven vs. nine) and (ii) the two beta sheets packed against each otherare connected by a disulfide bridge in the camel and llama domains, butnot in 10Fn3.

The three loops of ¹⁰Fn3 corresponding to the antigen-binding loops ofthe IgG heavy chain run between amino acid residues 21-31 (BC), 51-56(DE), and 76-88 (FG) (refer to FIG. 3 of U.S. Pat. No. 7,115,396, thecomplete contents of which are incorporated herein by reference). Thelengths of the BC and DE loops, 11 and 6 residues, respectively, fallwithin the narrow range of the corresponding antigen-recognition loopsfound in antibody heavy chains, that is, 7-10 and 4-8 residues,respectively. Accordingly, a CDR grafting strategy can be readilyemployed to introduce heavy chain CDR sequences into these domains.Additionally and/or alternatively, these two loops can be subjected tointroduction of genetic variability by any art-recognized method (e.g.,site-directed, look-through or other mutagenesis method, randomization,etc.) and, optionally, the resulting polypeptide may be subjected toselection for high antigen affinity. (Alternatively, introduction ofgenetic variability and/or selection procedures can be used to identifycompositions with lowered binding affinity and/or optimized propertiessuch as stability, toxicity, etc.) Through use of such methods, the BCand DE loops of fibronectin can be engineered to make contacts withantigens equivalent to the contacts of the corresponding CDR1 and CDR2domains in antibodies.

Unlike the BC and DE loops, the FG loop of ¹⁰Fn3 is 12 residues long,whereas the corresponding loop in antibody heavy chains ranges from 4-28residues. Accordingly, to optimize antigen binding, the FG loop of ¹⁰Fn3can be varied in length (e.g., via use of randomization and/or use ofpolypeptide linker sequences (which also can be randomized)) as well asin sequence to cover the CDR3 length range of 4-28 residues to obtainthe greatest possible flexibility and affinity in antigen binding.Indeed, for both those methods in which CDRs are directly grafted into afibronectin scaffold and those in which a native fibronectin scaffold isselected and/or optimized for binding of serum albumin (or other targetantigen), the lengths as well as the sequences of the CDR-like loops ofthe antibody mimics may be randomized during in vitro or in vivoaffinity maturation (as described in more detail below).

The tenth human fibronectin type III domain, ¹⁰Fn3, refolds rapidly evenat low temperature; its backbone conformation has been recovered within1 second at 5° C. Thermodynamic stability of ¹⁰Fn3 is high (ΔG_(n)=24kJ/mol=5.7 kcal/mol), correlating with its high melting temperature of110° C.

One of the physiological roles of ¹⁰Fn3 is as a subunit of fibronectin,a glycoprotein that exists in a soluble form in body fluids and in aninsoluble form in the extracellular matrix (Dickinson et al., J. Mol.Biol. 236:1079, 1994). A fibronectin monomer of 220-250 Kd contains 12type I modules, two type II modules, and 17 fibronectin type III modules(Potts and Campbell, Curr. Opin. Cell Biol. 6:648, 1994). Different typeIII modules are involved in the binding of fibronectin to integrins,heparin, and chondroitin sulfate. ¹⁰Fn3 was found to mediate celladhesion through an integrin-binding Arg-Gly-Asp (RGD) motif on one ofits exposed loops. Similar RGD motifs have been shown to be involved inintegrin binding by other proteins, such as fibrinogen, von Wellebrandfactor, and vitronectin (Hynes et al., Cell 69:11, 1992). No othermatrix- or cell-binding roles have been described for ¹⁰Fn3.

The observation that ¹⁰Fn3 has only slightly more adhesive activity thana short peptide containing RGD is consistent with the conclusion thatthe cell-binding activity of ¹⁰Fn3 is localized in the RGD peptiderather than distributed throughout the ¹⁰Fn3 structure (Baron et al.,Biochemistry 31:2068, 1992). The fact that ¹⁰Fn3 without the RGD motifis unlikely to bind to other plasma proteins or extracellular matrixmakes ¹⁰Fn3 a useful scaffold to replace antibodies. In addition, thepresence of ¹⁰Fn3 in natural fibrinogen in the bloodstream indicatesthat ¹⁰Fn3 itself is unlikely to be immunogenic in the organism oforigin.

In addition, it was shown that the ¹⁰Fn3 framework possesses exposedloop sequences tolerant of randomization, facilitating the generation ofdiverse pools of antibody mimics. This determination was made byexamining the flexibility of the ¹⁰Fn3 sequence. In particular, thehuman ¹⁰Fn3 sequence was aligned with the sequences of fibronectins fromother sources as well as sequences of related proteins, and the resultsof this alignment were mapped onto the three-dimensional structure ofthe human ¹⁰Fn3 domain. This alignment revealed that the majority ofconserved residues were found in the core of the beta sheet sandwich,whereas the highly variable residues were located along the edges of thebeta sheets, including the N- and C-termini, on the solvent-accessiblefaces of both beta sheets, and on three solvent-accessible loops thatserved as the hypervariable loops for affinity maturation of theantibody mimics. In view of these results, the randomization of thesethree loops was determined to be unlikely to have an adverse effect onthe overall fold or stability of the ¹⁰Fn3 framework itself.

For the human ¹⁰Fn3 sequence, this analysis indicated that, at aminimum, amino acids 1-9, 44-50, 61-54, 82-94 (edges of beta sheets);19, 21, 30-46 (even), 79-65 (odd) (solvent-accessible faces of both betasheets); 21-31, 51-56, 76-88 (CDR-like solvent-accessible loops); and14-16 and 36-45 (other solvent-accessible loops and beta turns) could berandomized to evolve new or improved compound-binding proteins. Inaddition, as discussed above, alterations in the lengths of one or moresolvent exposed loops could also be included in such directed evolutionmethods.

Alternatively, changes in the β-sheet sequences could also be used toevolve new proteins. These mutations change the scaffold and therebyindirectly alter loop structure(s). If this approach is taken, mutationsshould not saturate the sequence, but rather few mutations should beintroduced. Preferably, no more than between 3-20 changes should beintroduced to the β-sheet sequences by this approach.

Sequence variation can be introduced by any technique including, forexample, mutagenesis by Taq polymerase (Tindall and Kunkel, Biochemistry27:6008 (1988)), fragment recombination, or a combination thereof.Similarly, an increase of the structural diversity of libraries, forexample, by varying the length as well as the sequence of theCDR-presenting and/or CDR-like loops, or by structural redesign based onthe advantageous framework mutations found in selected pools, can beused to introduce further improvements in non-immunoglobulin scaffolds.

Fusion Proteins Comprising Fibronectin Scaffold Polypeptides

The fibronectin scaffold polypeptides described herein may be fused toother protein domains. For example, fibronectin scaffold polypeptidesidentified to bind human serum albumin can be fused with heavy chainsingle variable domains, or antigen binding fragments thereof, in orderto generate a dual-specific ligand of the invention comprising afibronectin-based serum albumin binding moiety. Fibronectin scaffoldpolypeptides additionally may be integrated with the human immuneresponse by fusing the constant region of an IgG (Fe) with a fibronectinscaffold polypeptide, such as an ¹⁰Fn3 module, preferably through theC-terminus of ¹⁰Fn3. The F_(c) in such a ¹⁰Fn3-F_(c) fusion moleculeactivates the complement component of the immune response and can serveto increase the therapeutic value of the engineered fibronectinpolypeptide. Similarly, a fusion between a fibronectin scaffoldpolypeptide, such as ¹⁰Fn3, and a complement protein, such as C1q, maybe used to target cells, and a fusion between a fibronectin scaffoldpolypeptide, such as ¹⁰Fn3, and a toxin may be used to specificallydestroy cells that carry a particular antigen. Any of these fusions maybe generated by standard techniques, for example, by expression of thefusion protein from a recombinant fusion gene constructed using publiclyavailable gene sequences and/or as otherwise described infra.

Scaffold Multimers

In addition to monomers, any of the fibronectin scaffold constructsdescribed herein may be generated as dimers or multimers of scaffolds asa means to increase the valency and thus the avidity of antigen (e.g.,serum albumin) binding. Such multimers may be generated through covalentbinding. For example, individual 10Fn3 modules may be bound by imitatingthe natural 8Fn3-9Fn3-10Fn3 C-to-N-terminus binding or by imitatingantibody dimers that are held together through their constant regions. A10Fn3-Fc construct may be exploited to design dimers of the generalscheme of 10Fn3-Fc::Fc-10Fn3. The bonds engineered into the Fc::Fcinterface may be covalent or non-covalent. In addition, dimerizing ormultimerizing partners other than Fc, such as other non-immunoglobulinscaffold moieties and/or immunoglobulin-based antigen-binding moieties,can be used in hybrids, such as 10Fn3 hybrids, to create such higherorder structures. Other examples of multimers include single variabledomains described herein.

In particular examples, covalently bonded multimers may be generated byconstructing fusion genes that encode the multimer or, alternatively, byengineering codons for cysteine residues into monomer sequences andallowing disulfide bond formation to occur between the expressionproducts. Non-covalently bonded multimers may also be generated by avariety of techniques. These include the introduction, into monomersequences, of codons corresponding to positively and/or negativelycharged residues and allowing interactions between these residues in theexpression products (and therefore between the monomers) to occur. Thisapproach may be simplified by taking advantage of charged residuesnaturally present in a monomer subunit, for example, the negativelycharged residues of fibronectin. Another means for generatingnon-covalently bonded compositions comprising fibronectin scaffoldpolypeptides is to introduce, into the monomer gene (for example, at theamino- or carboxy-termini), the coding sequences for proteins or proteindomains known to interact. Such proteins or protein domains includecoil-coil motifs, leucine zipper motifs, and any of the numerous proteinsubunits (or fragments thereof) known to direct formation of dimers orhigher order multimers.

Fibronectin-Like Molecules

Although ¹⁰Fn3 represents a preferred scaffold for the generation ofantibody mimics, other molecules may be substituted for ¹⁰Fn3 in themolecules described herein. These include, without limitation, humanfibronectin modules ¹Fn3-⁹Fn3 and ¹¹Fn3-¹⁷Fn3 as well as related Fn3modules from non-human animals and prokaryotes. In addition, Fn3 modulesfrom other proteins with sequence homology to ¹⁰Fn3, such as tenascinsand undulins, may also be used. Other exemplary scaffolds havingimmunoglobulin-like folds (but with sequences that are unrelated to theV_(H) domain) include N-cadherin, ICAM-2, titin, GCSF receptor, cytokinereceptor, glycosidase inhibitor, E-cadherin, and antibioticchromoprotein. Further domains with related structures may be derivedfrom myelin membrane adhesion molecule P0, CD8, CD4, CD2, class I MHC,T-cell antigen receptor, CD1, C2 and I-set domains of VCAM-1, I-setimmunoglobulin domain of myosin-binding protein C, I-set immunoglobulindomain of myosin-binding protein H, I-set immunoglobulin domain oftelokin, telikin, NCAM, twitchin, neuroglian, growth hormone receptor,erythropoietin receptor, prolactin receptor, GC-SF receptor,interferon-gamma receptor, β-galactosidase/glucuronidase,β-glucuronidase, and transglutaminase. Alternatively, any other proteinthat includes one or more immunoglobulin-like folds may be utilized.Such proteins may be identified, for example, using the program SCOP(Murzin et al., J. Mol. Biol. 247:536 (1995); Lo Conte et al., NucleicAcids Res. 25:257 (2000).

Generally, any molecule that exhibits a structural relatedness to theV_(H) domain (as identified, for example, using the SCOP computerprogram above) can be utilized as a non-immunoglobulin scaffold. Suchmolecules may, like fibronectin, include three loops at the N-terminalpole of the molecule and three loops at the C-terminal pole, each ofwhich may be randomized to create diverse libraries; alternatively,larger domains may be utilized, having larger numbers of loops, as longas a number of such surface randomizable loops are positioned closelyenough in space that they can participate in antigen binding. Examplesof polypeptides possessing more than three loops positioned close toeach other include T-cell antigen receptor and superoxide dismutase,which each have four loops that can be randomized; and an Fn3 dimer,tissue factor domains, and cytokine receptor domains, each of which havethree sets of two similar domains where three randomizable loops arepart of the two domains (bringing the total number of loops to six).

In yet another alternative, any protein having variable loops positionedclose enough in space may be utilized for candidate binding proteinproduction. For example, large proteins having spatially related,solvent accessible loops may be used, even if unrelated structurally toan immunoglobulin-like fold. Exemplary proteins include, withoutlimitation, cytochrome F, green fluorescent protein, GroEL, andthaumatin. The loops displayed by these proteins may be randomized andsuperior binders selected from a randomized library as described herein,e.g. Example 1. Because of their size, molecules may be obtained thatexhibit an antigen binding surface considerably larger than that foundin an antibody-antigen interaction. Other useful scaffolds of this typemay also be identified using the program SCOP (Murzin et al., J. Mol.Biol. 247: 536 (1995)) to browse among candidate proteins havingnumerous loops, particularly loops positioned among parallel beta sheetsor a number of alpha-helices.

Modules from different organisms and parent proteins may be mostappropriate for different applications. For example, in designing afibronectin scaffold polypeptide of the invention, it may be mostdesirable to generate that protein from a fibronectin orfibronectin-like molecule native to the organism for which a therapeuticis intended. In contrast, the organism of origin is less important oreven irrelevant for fibronectin scaffolds that are to be used for invitro applications, such as diagnostics, or as research reagents.

For any of these molecules, libraries may be generated and used toselect binding proteins by any of the methods described herein.

Directed Evolution of Scaffold-Based Binding Proteins

The non-immunoglobulin scaffolds described herein may be used in anytechnique for evolving new or improved binding proteins. In oneparticular example, the target of binding (e.g., serum albumin) isimmobilized on a solid support, such as a column resin or microtiterplate well, and the target contacted with a library of candidatenon-immunoglobulin scaffold-based binding proteins. Such a library mayconsist of fibronectin scaffold clones, such as ¹⁰Fn3 clones constructedfrom the native (wild type) ¹⁰Fn3 scaffold through randomization of thesequence and/or the length of the ¹⁰Fn3 CDR-like loops. If desired, thislibrary may be an RNA-protein fusion library generated, for example, bythe techniques described in Szostak et al., U.S. Ser. No. 09/007,005 andSer. No. 09/247,190; Szostak et al., WO98/31700; and Roberts & Szostak,Proc. Natl. Acad. Sci. USA (1997) vol. 94, p. 12297-12302.Alternatively, it may be a DNA-protein library (for example, asdescribed in Lohse, DNA-Protein Fusions and Uses Thereof, U.S. Ser. No.60/110,549, U.S. Ser. No. 09/459,190, and WO 00/32823). The fusionlibrary is incubated with the immobilized target, the support is washedto remove non-specific binders, and the tightest binders are elutedunder very stringent conditions and subjected to PCR to recover thesequence information or to create a new library of binders which may beused to repeat the selection process, with or without furthermutagenesis of the sequence. A number of rounds of selection may beperformed until binders of sufficient affinity for the antigen (e.g.,serum albumin) are obtained.

In one particular example, the ¹⁰Fn3 scaffold may be used as theselection target. For example, if a protein is required that binds aspecific peptide sequence (e.g., serum albumin) presented in a tenresidue loop, a single ¹⁰Fn3 clone is constructed in which one of itsloops has been set to the length of ten and to the desired sequence. Thenew clone is expressed in vivo and purified, and then immobilized on asolid support. An RNA-protein fusion library based on an appropriatescaffold is then allowed to interact with the support, which is thenwashed, and desired molecules eluted and re-selected as described above.

Similarly, the scaffolds described herein, for example, the ¹⁰Fn3scaffold, may be used to find natural proteins that interact with thepeptide sequence displayed by the scaffold, for example, in an ¹⁰Fn3loop. The scaffold protein, such as the ¹⁰Fn3 protein, is immobilized asdescribed above, and an RNA-protein fusion library is screened forbinders to the displayed loop. The binders are enriched through multiplerounds of selection and identified by DNA sequencing.

In addition, in the above approaches, although RNA-protein librariesrepresent exemplary libraries for directed evolution, any type ofscaffold-based library may be used in the selection methods of theinvention.

Use of Fibronectin Scaffold Polypeptides

The fibronectin scaffold polypeptides described herein may be evolved tobind serum albumin or any antigen of interest. Such fibronectin scaffoldproteins have thermodynamic properties superior to those of naturalantibodies and can be evolved rapidly in vitro. Accordingly, thesefibronectin scaffold polypeptides may be employed to produce bindingdomains for use in the research, therapeutic, and diagnostic fields.

Mutagenic Affinity Maturation

The selections described herein may also be combined with mutagenesisafter all or a subset of the selection steps to further increase librarydiversity. Methods of affinity maturation may employ, e.g., error-pronePCR (Cadwell and Joyce, PCR Methods Appl 2:28 (1992)) or alternativeforms of random mutagenesis, NNK mutagenesis as described infra,look-through mutagenesis (wherein CDR-grafted fibronectin scaffoldpolypeptides are engineered to optimize antigen binding through use ofnaturally-occurring CDR diversity—refer, e.g., to WO 06/023144,incorporated herein by reference), and/or other art-recognized mutagenicapproach for creating polypeptide diversity, that is combined with oneor more rounds of selection for antigen-binding affinity.

Any of the scaffold proteins described infra may be combined with oneanother for use, e.g., in the dual-specific ligand compositions of thepresent invention. For example, CDRs may be grafted on to a CTLA-4scaffold and used together with antibody VH or VL domains to form amultivalent ligand. Likewise, fibronectin, lipocalin, affibodies, andother scaffolds may be combined.

All publications mentioned in the present specification, and referencescited in said publications, are herein incorporated by reference.Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

Annex 1; polypeptides which enhance half-life in vivo.

Alpha-1 Glycoprotein (Orosomucoid) (AAG) Alpha-1 Antichyromotrypsin(ACT) Alpha-1 Antitrypsin (AAT) Alpha-1 Microglobulin (Protein HC) (AIM)Alpha-2 Macroglobulin (A2M) Antithrombin III (AT III) Apolipoprotein A-1(Apo A-1) Apoliprotein B (Apo B)

Beta-2-microglobulin (B2M)

Ceruloplasmin (Cp) Complement Component (C3) Complement Component (C4)C1 Esterase Inhibitor (C1 INH) C-Reactive Protein (CRP) Cystatin C (CysC) Ferritin (FER) Fibrinogen (FIB) Fibronectin (FN) Haptoglobin (Hp)Hemopexin (HPX) Immunoglobulin A (IgA) Immunoglobulin D (IgD)Immunoglobulin E (IgE) Immunoglobulin G (IgG) Immunoglobulin M (IgM)

Immunoglobulin Light Chains (kapa/lambda)

Lipoprotein(a) [Lp(a)]

Mannose-bindign protein (MBP)

Myoglobin (Myo) Plasminogen (PSM) Prealbumin (Transthyretin) (PAL)

Retinol-binding protein (RBP)

Rheomatoid Factor (RF) Serum Amyloid A (SAA)

Soluble Tranferrin Receptor (sTfR)

Transferrin (Tf)

Annex 2 Pairing Therapeutic relevant references. TNF TGF-b and TNF wheninjected into the ankle joint of collagen induced ALPHA/TGF-β arthritismodel significantly enhanced joint inflammation. In non-collagenchallenged mice there was no effect. TNF ALPHA/IL-1 TNF and IL-1synergize in the pathology of uveitis. TNF and IL-1 synergize in thepathology of malaria (hypoglycaemia, NO). TNF and IL-1 synergize in theinduction of polymorphonuclear (PMN) cells migration in inflammation.IL-1 and TNF synergize to induce PMN infiltration into the peritoneum.IL-1 and TNF synergize to induce the secretion of IL-1 by endothelialcells. Important in inflammation. IL-1 or TNF alone induced somecellular infiltration into knee synovium. IL-1 induced PMNs, TNF -monocytes. Together they induced a more severe infiltration due toincreased PMNs. Circulating myocardial depressant substance (present insepsis) is low levels of IL-1 and TNFacting synergistically. TNFALPHA/IL-2 Most relating to synergisitic activation of killer T-cells.TNF ALPHA/IL-3 Synergy of interleukin 3 and tumor necrosis factor alphain stimulating clonal growth of acute myelogenous leukemia blasts is theresult of induction of secondary hematopoietic cytokines by tumornecrosis factor alpha. Cancer Res. 1992 Apr. 15; 52(8): 2197-201. TNFALPHA/IL-4 IL-4 and TNF synergize to induce VCAM expression onendothelial cells. Implied to have a role in asthma. Same for synovium -implicated in RA. TNF and IL-4 synergize to induce IL-6 expression inkeratinocytes. Sustained elevated levels of VCAM-1 in culturedfibroblast-like synoviocytes can be achieved by TNF-alpha in combinationwith either IL- 4 or IL-13 through increased mRNA stability. Am JPathol. 1999 April; 154(4): 1149-58 TNF ALPHA/IL-5 Relationship betweenthe tumor necrosis factor system and the serum interleukin-4,interleukin-5, interleukin-8, eosinophil cationic protein, andimmunoglobulin E levels in the bronchial hyperreactivity of adults andtheir children. Allergy Asthma Proc. 2003 March-April; 24(2): 111-8. TNFALPHA/IL-6 TNF and IL-6 are potent growth factors for OH-2, a novelhuman myeloma cell line. Eur J Haematol. 1994 July; 53(1): 31-7. TNFALPHA/IL-8 TNF and IL-8 synergized with PMNs to activate platelets.Implicated in Acute Respiratory Distress Syndrome. See IL-5/TNF(asthma). Synergism between interleukin-8 and tumor necrosisfactor-alpha for neutrophil-mediated platelet activation. Eur CytokineNetw. 1994 September-October; 5(5): 455-60. (adult respiratory distresssyndrome (ARDS)) TNF ALPHA/IL-9 TNF ALPHA/IL-10 IL-10 induces andsynergizes with TNF in the induction of HIV expression in chronicallyinfected T-cells. TNF ALPHA/IL-11 Cytokines synergistically induceosteoclast differentiation: support by immortalized or normal calvarialcells. Am J Physiol Cell Physiol. 2002 September; 283(3): C679-87. (Boneloss) TNF ALPHA/IL-12 TNF ALPHA/IL-13 Sustained elevated levels ofVCAM-1 in cultured fibroblast-like synoviocytes can be achieved byTNF-alpha in combination with either IL- 4 or IL-13 through increasedmRNA stability. Am J Pathol. 1999 April; 154(4): 1149-58. Interleukin-13and tumour necrosis factor-alpha synergistically induce eotaxinproduction in human nasal fibroblasts. Clin Exp Allergy. 2000 March;30(3): 348-55. Interleukin-13 and tumour necrosis factor-alphasynergistically induce eotaxin production in human nasal fibroblasts.Clin Exp Allergy. 2000 March; 30(3): 348-55 (allergic inflammation)Implications of serum TNF-beta and IL-13 in the treatment response ofchildhood nephrotic syndrome. Cytokine. 2003 Feb. 7; 21(3): 155-9. TNFALPHA/IL-14 Effects of inhaled tumour necrosis factor alpha in subjectswith mild asthma. Thorax. 2002 September; 57(9): 774-8. TNF ALPHA/IL-15Effects of inhaled tumour necrosis factor alpha in subjects with mildasthma. Thorax. 2002 September; 57(9): 774-8. TNF ALPHA/IL-16 Tumornecrosis factor-alpha-induced synthesis of interleukin-16 in airwayepithelial cells: priming for serotonin stimulation. Am J Respir CellMol Biol. 2003 March; 28(3): 354-62. (airway inflammation) Correlationof circulating interleukin 16 with proinflammatory cytokines in patientswith rheumatoid arthritis. Rheumatology (Oxford). 2001 April; 40(4):474-5. No abstract available. Interleukin 16 is up-regulated in Crohn'sdisease and participates in TNBS colitis in mice. Gastroenterology. 2000October; 119(4): 972-82. TNF ALPHA/IL-17 Inhibition of interleukin-17prevents the development of arthritis in vaccinated mice challenged withBorrelia burgdorferi. Infect Immun. 2003 June; 71(6): 3437-42.Interleukin 17 synergises with tumour necrosis factor alpha to inducecartilage destruction in vitro. Ann Rheum Dis. 2002 October; 61(10):870-6. A role of GM-CSF in the accumulation of neutrophils in theairways caused by IL-17 and TNF-alpha. Eur Respir J. 2003 March; 21(3):387-93. (Airway inflammation) Abstract Interleukin-1, tumor necrosisfactor alpha, and interleukin-17 synergistically up-regulate nitricoxide and prostaglandin E2 production in explants of humanosteoarthritic knee menisci. Arthritis Rheum. 2001 September; 44(9):2078-83. TNF ALPHA/IL-18 Association of interleukin-18 expression withenhanced levels of both interleukin-1beta and tumor necrosis factoralpha in knee synovial tissue of patients with rheumatoid arthritis.Arthritis Rheum. 2003 February; 48(2): 339- 47. Abstract Elevated levelsof interleukin-18 and tumor necrosis factor-alpha in serum of patientswith type 2 diabetes mellitus: relationship with diabetic nephropathy.Metabolism. 2003 May; 52(5): 605-8. TNF ALPHA/IL-19 Abstract IL-19induces production of IL-6 and TNF-alpha and results in cell apoptosisthrough TNF-alpha. J Immunol. 2002 Oct. 15; 169(8): 4288- 97. TNFALPHA/IL-20 Abstract Cytokines: IL-20 - a new effector in skininflammation. Curr Biol. 2001 Jul. 10; 11(13): R531-4 TNF Inflammationand coagulation: implications for the septic patient. ClinALPHA/Complement Infect Dis. 2003 May 15; 36(10): 1259-65. Epub 2003 May8. Review. TNF MHC induction in the brain. ALPHA/IFN-γ Synergize inanti-viral response/IFN-β induction. Neutrophil activation/respiratoryburst. Endothelial cell activation Toxicities noted when patientstreated with TNF/IFN-γ as anti-viral therapy Fractalkine expression byhuman astrocytes. Many papers on inflammatory responses - i.e. LPS, alsomacrophage activation. Anti-TNF and anti-IFN-γ synergize to protect micefrom lethal endotoxemia. TGF-β/IL-1 Prostaglndin synthesis byosteoblasts IL-6 production by intestinal epithelial cells (inflammationmodel) Stimulates IL-11 and IL-6 in lung fibroblasts (inflammationmodel) IL-6 and IL-8 production in the retina TGF-β/IL-6 Chondrocarcomaproliferation IL-1/IL-2 B-cell activation LAK cell activation T-cellactivation IL-1 synergy with IL-2 in the generation of lymphokineactivated killer cells is mediated by TNF-alpha and beta (lymphotoxin).Cytokine. 1992 November; 4(6): 479-87. IL-1/IL-3 IL-1/IL-4 B-cellactivation IL-4 induces IL-1 expression in endothelial cell activation.IL-1/IL-5 IL-1/IL-6 B cell activation T cell activation (can replaceaccessory cells) IL-1 induces IL-6 expression C3 and serum amyloidexpression (acute phase response) HIV expression Cartilage collagenbreaKdown. IL-1/IL-7 IL-7 is requisite for IL-1-induced thymocyteproliferation. Involvement of IL-7 in the synergistic effects ofgranulocyte-macrophage colony- stimulating factor or tumor necrosisfactor with IL-1. J Immunol. 1992 Jan. 1; 148(1): 99-105. IL-1/IL-8IL-1/IL-10 IL-1/IL-11 Cytokines synergistically induce osteoclastdifferentiation: support by immortalized or normal calvarial cells. Am JPhysiol Cell Physiol. 2002 September; 283(3): C679-87. (Bone loss)IL-1/IL-16 Correlation of circulating interleukin 16 withproinflammatory cytokines in patients with rheumatoid arthritis.Rheumatology (Oxford). 2001 April; 40(4): 474-5. No abstract available.IL-1/IL-17 Inhibition of interleukin-17 prevents the development ofarthritis in vaccinated mice challenged with Borrelia burgdorferi.Infect Immun. 2003 June; 71(6): 3437-42. Contribution of interleukin 17to human cartilage degradation and synovial inflammation inosteoarthritis. Osteoarthritis Cartilage. 2002 October; 10(10): 799-807.Abstract Interleukin-1, tumor necrosis factor alpha, and interleukin-17synergistically up-regulate nitric oxide and prostaglandin E2 productionin explants of human osteoarthritic knee menisci. Arthritis Rheum. 2001September; 44(9): 2078-83. IL-1/IL-18 Association of interleukin-18expression with enhanced levels of both interleukin-1beta and tumornecrosis factor alpha in knee synovial tissue of patients withrheumatoid arthritis. Arthritis Rheum. 2003 February; 48(2): 339-47.IL-1/IFN-g IL-2/IL-3 T-cell proliferation B cell proliferation IL-2/IL-4B-cell proliferation T-cell proliferation (selectively inducingactivation of CD8 and NK lymphocytes)IL-2R beta agonist P1-30 acts insynergy with IL-2, IL-4, IL-9, and IL-15: biological and moleculareffects. J Immunol. 2000 Oct. 15; 165(8): 4312-8. IL-2/IL-5 B-cellproliferation/Ig secretion IL-5 induces IL-2 receptors on B-cellsIL-2/IL-6 Development of cytotoxic T-cells IL-2/IL-7 IL-2/IL-9 SeeIL-2/IL-4 (NK-cells) IL-2/IL-10 B-cell activation IL-2/IL-12 IL-12synergizes with IL-2 to induce lymphokine-activated cytotoxicity andperforin and granzyme gene expression in fresh human NK cells. CellImmunol. 1995 Oct. 1; 165(1): 33-43. (T-cell activation) IL-2/IL-15 SeeIL-2/IL-4 (NK cells) (T cell activation and proliferation) IL-15 andIL-2: a matter of life and death for T cells in vivo. Nat Med. 2001January; 7(1): 114-8. IL-2/IL-16 Synergistic activation of CD4+ T cellsby IL-16 and IL-2. J Immunol. 1998 Mar. 1; 160(5): 2115-20. IL-2/IL-17Evidence for the early involvement of interleukin 17 in human andexperimental renal allograft rejection. J Pathol. 2002 July; 197(3):322-32. IL-2/IL-18 Interleukin 18 (IL-18) in synergy with IL-2 induceslethal lung injury in mice: a potential role for cytokines, chemokines,and natural killer cells in the pathogenesis of interstitial pneumonia.Blood. 2002 Feb. 15; 99(4): 1289- 98. IL-2/TGF-β Control of CD4 effectorfate: transforming growth factor beta 1 and interleukin 2 synergize toprevent apoptosis and promote effector expansion. J Exp Med. 1995 Sep.1; 182(3): 699-709. IL-2/IFN-γ Ig secretion by B-cells IL-2 inducesIFN-γ expression by T-cells IL-2/IFN-α/β None IL-3/IL-4 Synergize inmast cell growth Synergistic effects of IL-4 and either GM-CSF or IL-3on the induction of CD23 expression by human monocytes: regulatoryeffects of IFN-alpha and IFN-gamma. Cytokine. 1994 July; 6(4): 407-13.IL-3/IL-5 IL-3/IL-6 IL-3/IFN-γ IL-4 and IFN-gamma synergisticallyincrease total polymeric IgA receptor levels in human intestinalepithelial cells. Role of protein tyrosine kinases. J Immunol. 1996 Jun.15; 156(12): 4807-14. IL-3/GM-CSF Differential regulation of humaneosinophil IL-3, IL-5, and GM-CSF receptor alpha-chain expression bycytokines: IL-3, IL-5, and GM-CSF down-regulate IL-5 receptor alphaexpression with loss of IL-5 responsiveness, but up-regulate IL-3receptor alpha expression. J Immunol. 2003 Jun. 1; 170(11): 5359-66.(allergic inflammation) IL-4/IL-2 IL-4 synergistically enhances bothIL-2- and IL-12-induced IFN-{gamma} expression in murine NK cells.Blood. 2003 Mar. 13 [Epub ahead of print] IL-4/IL-5 Enhanced mast cellhistamine etc. secretion in response to IgE A Th2-like cytokine responseis involved in bullous pemphigoid. the role of IL-4 and IL-5 in thepathogenesis of the disease. Int J Immunopathol Pharmacol. 1999May-August; 12(2): 55-61. IL-4/IL-6 IL-4/IL-10 IL-4/IL-11 Synergisticinteractions between interleukin-11 and interleukin-4 in support ofproliferation of primitive hematopoietic progenitors of mice. Blood.1991 Sep. 15; 78(6): 1448-51. IL-4/IL-12 Synergistic effects of IL-4 andIL-18 on IL-12-dependent IFN-gamma production by dendritic cells. JImmunol. 2000 Jan. 1; 164(1): 64-71. (increase Th1/Th2 differentiation)IL-4 synergistically enhances both IL-2- and IL-12-induced IFN-{gamma}expression in murine NK cells. Blood. 2003 Mar. 13 [Epub ahead of print]IL-4/IL-13 Abstract Interleukin-4 and interleukin-13 signalingconnections maps. Science. 2003 Jun. 6; 300(5625): 1527-8. (allergy,asthma) Inhibition of the IL-4/IL-13 receptor system prevents allergicsensitization without affecting established allergy in a mouse model forallergic asthma. J Allergy Clin Immunol. 2003 June; 111(6): 1361-1369.IL-4/IL-16 (asthma) Interleukin (IL)-4/IL-9 and exogenous IL-16 induceIL-16 production by BEAS-2B cells, a bronchial epithelial cell line.Cell Immunol. 2001 Feb. 1; 207(2): 75-80 IL-4/IL-17 Interleukin (IL)-4and IL-17 synergistically stimulate IL-6 secretion in human colonicmyofibroblasts. Int J Mol Med. 2002 November; 10(5): 631-4. (Gutinflammation) IL-4/IL-24 IL-24 is expressed by rat and humanmacrophages. Immunobiology. 2002 July; 205(3): 321-34. IL-4/IL-25Abstract New IL-17 family members promote Th1 or Th2 responses in thelung: in vivo function of the novel cytokine IL-25. J Immunol. 2002 Jul.1; 169(1): 443-53. (allergic inflammation) Abstract Mast cells produceinterleukin-25 upon Fcepsilon RI-mediated activation. Blood. 2003 May 1;101(9): 3594-6. Epub 2003 Jan. 2. (allergic inflammation) IL-4/IFN-γAbstract Interleukin 4 induces interleukin 6 production by endothelialcells: synergy with interferon-gamma. Eur J Immunol. 1991 January;21(1): 97-101. IL-4/SCF Regulation of human intestinal mast cells bystem cell factor and IL-4. Immunol Rev. 2001 February; 179: 57-60.Review. IL-5/IL-3 Differential regulation of human eosinophil IL-3,IL-5, and GM-CSF receptor alpha-chain expression by cytokines: IL-3,IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with lossof IL-5 responsiveness, but up-regulate IL-3 receptor alpha expression.J Immunol. 2003 Jun. 1; 170(11): 5359-66. (Allergic inflammation seeabstract) IL-5/IL-6 IL-5/IL-13 Inhibition of allergic airwaysinflammation and airway hyperresponsiveness in mice by dexamethasone:role of eosinophils, IL-5, eotaxin, and IL-13. J Allergy Clin Immunol.2003 May; 111(5): 1049-61. IL-5/IL-17 Interleukin-17 orchestrates thegranulocyte influx into airways after allergen inhalation in a mousemodel of allergic asthma. Am J Respir Cell Mol Biol. 2003 January;28(1): 42-50. IL-5/IL-25 Abstract New IL-17 family members promote Th1or Th2 responses in the lung: in vivo function of the novel cytokineIL-25. J Immunol. 2002 Jul. 1; 169(1): 443-53. (allergic inflammation)Abstract Mast cells produce interleukin-25 upon Fcepsilon RI-mediatedactivation. Blood. 2003 May 1; 101(9): 3594-6. Epub 2003 Jan. 2.(allergic inflammation) IL-5/IFN-γ IL-5/GM-CSF Differential regulationof human eosinophil IL-3, IL-5, and GM-CSF receptor alpha-chainexpression by cytokines: IL-3, IL-5, and GM-CSF down-regulate IL-5receptor alpha expression with loss of IL-5 responsiveness, butup-regulate IL-3 receptor alpha expression. J Immunol. 2003 Jun. 1;170(11): 5359-66. (Allergic inflammation) IL-6/IL-10 IL-6/IL-11IL-6/IL-16 Interleukin-16 stimulates the expression and production ofpro- inflammatory cytokines by human monocytes. Immunology. 2000 May;100(1): 63-9. IL-6/IL-17 Stimulation of airway mucin gene expression byinterleukin (IL)-17 through IL-6 paracrine/autocrine loop. J Biol Chem.2003 May 9; 278(19): 17036-43. Epub 2003 Mar. 6. (airway inflammation,asthma) IL-6/IL-19 Abstract IL-19 induces production of IL-6 andTNF-alpha and results in cell apoptosis through TNF-alpha. J Immunol.2002 Oct. 15; 169(8): 4288- 97. IL-6/IFN-g IL-7/IL-2 Interleukin 7worsens graft-versus-host disease. Blood. 2002 Oct. 1; 100(7): 2642-9.IL-7/IL-12 Synergistic effects of IL-7 and IL-12 on human T cellactivation. J Immunol. 1995 May 15; 154(10): 5093-102. IL-7/IL-15Interleukin-7 and interleukin-15 regulate the expression of the bcl-2and c- myb genes in cutaneous T-cell lymphoma cells. Blood. 2001 Nov. 1;98(9): 2778-83. (growth factor) IL-8/IL-11 Abnormal production ofinterleukin (IL)-11 and IL-8 in polycythaemia vera. Cytokine. 2002 Nov.21; 20(4): 178-83. IL-8/IL-17 The Role of IL-17 in Joint Destruction.Drug News Perspect. 2002 January; 15(1): 17-23. (arthritis) AbstractInterleukin-17 stimulates the expression of interleukin-8, growth-related oncogene-alpha, and granulocyte-colony-stimulating factor byhuman airway epithelial cells. Am J Respir Cell Mol Biol. 2002 June;26(6): 748-53. (airway inflammation) IL-8/GSF Interleukin-8: anautocrine/paracrine growth factor for human hematopoietic progenitorsacting in synergy with colony stimulating factor- 1 to promotemonocyte-macrophage growth and differentiation. Exp Hematol. 1999January; 27(1): 28-36. IL-8/VGEF Intracavitary VEGF, bFGF, IL-8, IL-12levels in primary and recurrent malignant glioma. J Neurooncol. 2003May; 62(3): 297-303. IL-9/IL-4 Anti-interleukin-9 antibody treatmentinhibits airway inflammation and hyperreactivity in mouse asthma model.Am J Respir Crit Care Med. 2002 Aug. 1; 166(3): 409-16. IL-9/IL-5Pulmonary overexpression of IL-9 induces Th2 cytokine expression,leading to immune pathology. J Clin Invest. 2002 January; 109(1): 29-39.Th2 cytokines and asthma. Interleukin-9 as a therapeutic target forasthma. Respir Res. 2001; 2(2): 80-4. Epub 2001 Feb. 15. Review.Abstract Interleukin-9 enhances interleukin-5 receptor expression,differentiation, and survival of human eosinophils. Blood. 2000 Sep. 15;96(6): 2163-71 (asthma) IL-9/IL-13 Anti-interleukin-9 antibody treatmentinhibits airway inflammation and hyperreactivity in mouse asthma model.Am J Respir Crit Care Med. 2002 Aug. 1; 166(3): 409-16. Direct effectsof interleukin-13 on epithelial cells cause airway hyperreactivity andmucus overproduction in asthma. Nat Med. 2002 August; 8(8): 885-9.IL-9/IL-16 See IL-4/IL-16 IL-10/IL-2 The interplay of interleukin-10(IL-10) and interleukin-2 (IL-2) in humoral immune responses: IL-10synergizes with IL-2 to enhance responses of human B lymphocytes in amechanism which is different from upregulation of CD25 expression. CellImmunol. 1994 September; 157(2): 478-88. IL-10/IL-12 IL-10/TGF-β IL-10and TGF-beta cooperate in the regulatory T cell response to mucosalallergens in normal immunity and specific immunotherapy. Eur J Immunol.2003 May; 33(5): 1205-14. IL-10/IFN-γ IL-11/IL-6 Interleukin-6 andinterleukin-11 support human osteoclast formation by a RANKL-independentmechanism. Bone. 2003 January; 32(1): 1-7. (bone resorption ininflammation) IL-11/IL-17 Polarized in vivo expression of IL-11 andIL-17 between acute and chronic skin lesions. J Allergy Clin Immunol.2003 April; 111(4): 875-81. (allergic dermatitis) IL-17 promotes boneerosion in murine collagen-induced arthritis through loss of thereceptor activator of NF-kappa B ligand/osteoprotegerin balance. JImmunol. 2003 Mar. 1; 170(5): 2655-62. IL-11/TGF-β Polarized in vivoexpression of IL-11 and IL-17 between acute and chronic skin lesions. JAllergy Clin lmmunol. 2003 April; 111(4): 875-81. (allergic dermatitis)IL-12/IL-13 Relationship of Interleukin-12 and Interleukin-13 imbalancewith class- specific rheumatoid factors and anticardiolipin antibodiesin systemic lupus erythematosus. Clin Rheumatol. 2003 May; 22(2):107-11. IL-12/IL-17 Upregulation of interleukin-12 and -17 in activeinflammatory bowel disease. Scand J Gastroenterol. 2003 February; 38(2):180-5. IL-12/IL-18 Synergistic proliferation and activation of naturalkiller cells by interleukin 12 and interleukin 18. Cytokine. 1999November; 11(11): 822-30. Inflammatory Liver Steatosis Caused by IL-12and IL-18. J Interferon Cytokine Res. 2003 March; 23(3): 155-62.IL-12/IL-23 nterleukin-23 rather than interleukin-12 is the criticalcytokine for autoimmune inflammation of the brain. Nature. 2003 Feb. 13;421(6924): 744-8. Abstract A unique role for IL-23 in promoting cellularimmunity. J Leukoc Biol. 2003 January; 73(1): 49-56. Review. IL-12/IL-27Abstract IL-27, a heterodimeric cytokine composed of EBI3 and p28protein, induces proliferation of naive CD4(+) T cells. Immunity. 2002June; 16(6): 779-90. IL-12/IFN-γ IL-12 induces IFN-γ expression by B andT-cells as part of immune stimulation. IL-13/IL-5 See IL-5/IL-13IL-13/IL-25 Abstract New IL-17 family members promote Th1 or Th2responses in the lung: in vivo function of the novel cytokine IL-25. JImmunol. 2002 Jul. 1; 169(1): 443-53. (allergic inflammation) AbstractMast cells produce interleukin-25 upon Fcepsilon RI-mediated activation.Blood. 2003 May 1; 101(9): 3594-6. Epub 2003 Jan. 2. (allergicinflammation) IL-15/IL-13 Differential expression of interleukins(IL)-13 and IL-15 in ectopic and eutopic endometrium of women withendometriosis and normal fertile women. Am J Reprod Immunol. 2003February; 49(2): 75-83. IL-15/IL-16 IL-15 and IL-16 overexpression incutaneous T-cell lymphomas: stage- dependent increase in mycosisfungoides progression. Exp Dermatol. 2000 August; 9(4): 248-51.IL-15/IL-17 Abstract IL-17, produced by lymphocytes and neutrophils, isnecessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as apossible trigger. J Immunol. 2003 Feb. 15; 170(4): 2106-12. (airwayinflammation) IL-15/IL-21 IL-21 in Synergy with IL-15 or IL-18 EnhancesIFN-gamma Production in Human NK and T Cells. J Immunol. 2003 Jun. 1;170(11): 5464-9. IL-17/IL-23 Interleukin-23 promotes a distinct CD4 Tcell activation state characterized by the production of interleukin-17.J Biol Chem. 2003 Jan. 17; 278(3): 1910-4. Epub 2002 Nov. 3 IL-17/TGF-βPolarized in vivo expression of IL-11 and IL-17 between acute andchronic skin lesions. J Allergy Clin Immunol. 2003 April; 111(4):875-81. (allergic dermatitis) IL-18/IL-12 Synergistic proliferation andactivation of natural killer cells by interleukin 12 and interleukin 18.Cytokine. 1999 November; 11(11): 822-30. Abstract Inhibition of in vitroimmunoglobulin production by IL-12 in murine chronic graft-vs.-hostdisease: synergism with IL-18. Eur J Immunol. 1998 June; 28(6): 2017-24.IL-18/IL-21 IL-21 in Synergy with IL-15 or IL-18 Enhances IFN-gammaProduction in Human NK and T Cells. J Immunol. 2003 Jun. 1; 170(11):5464-9. IL-18/TGF-β Interleukin 18 and transforming growth factor beta1in the serum of patients with Graves' ophthalmopathy treated withcorticosteroids. Int Immunopharmacol. 2003 April; 3(4): 549-52.IL-18/IFN-γ Anti-TNF Synergistic therapeutic effect in DBA/1 arthriticmice. ALPHA/anti-CD4

Annex 3: Oncology combinations Target Disease Pair with CD89* Use ascytotoxic cell recruiter all CD19 B cell lymphomas HLA-DR CD5 HLA-DR Bcell lymphomas CD89 CD19 CD5 CD38 Multiple myeloma CD138 CD56 HLA-DRCD138 Multiple myeloma CD38 CD56 HLA-DR CD138 Lung cancer CD56 CEA CD33Acute myelod lymphoma CD34 HLA-DR CD56 Lung cancer CD138 CEA CEA Pancarcinoma MET receptor VEGF Pan carcinoma MET receptor VEGF Pancarcinoma MET receptor receptor IL-13 Asthma/pulmonary IL-4 inflammationIL-5 Eotaxin(s) MDC TARC TNFα IL-9 EGFR CD40L IL-25 MCP-1 TGFβ IL-4Asthma IL-13 IL-5 Eotaxin(s) MDC TARC TNFα IL-9 EGFR CD40L IL-25 MCP-1TGFβ Eotaxin Asthma IL-5 Eotaxin-2 Eotaxin-3 EGFR cancer HER2/neu HER3HER4 HER2 cancer HER3 HER4 TNFR1 RA/Crohn's disease IL-1R IL-6R IL-18RTNFα RA/Crohn's disease IL-1α/β IL-6 IL-18 ICAM-1 IL-15 IL-17 IL-1RRA/Crohn's disease IL-6R IL-18R IL-18R RA/Crohn's disease IL-6R

Annex 4 Data Summary Equilibrium dissocation ND50 for cell based TARGETdAb constant (Kd = Koff/Kon) Koff IC50 for ligand assay neutralisn assayTAR1 TAR1 300 nM to 5 pM 5 × 10⁻¹ to 1 × 10⁻⁷ 500 nM to 100 pM 500 nM to50 pM monomers (ie, 3 × 10⁻⁷ to 5 × 10⁻¹²), preferably 50 nM to 20 pMTAR1 dimers As TAR1 monomer As TAR1 monomer As TAR1 monomer As TAR1monomer TAR1 trimers As TAR1 monomer As TAR1 monomer As TAR1 monomer AsTAR1 monomer TAR1-5 TAR1-27 TAR1-5-19 30 nM monomer TAR1-5-19 With(Gly₄Ser)₃ linker = 20 nm = 30 nM homodimer With (Gly₄Ser)₅ linker = 2nm With (Gly₄Ser)₇ linker = 10 nm In Fab format = 1 nM = 3 nM = 15 nMTAR1-5-19 With (Gly₄Ser)_(n) linker heterodimers TAR1-5-19 d2 = 2 nMTAR1-5-19 d3 = 8 nM TAR1-5-19 d4 = 2-5 nM = 12 nM TAR1-5-19 d5 = 8 nM =10 nM In Fab format TAR1-5-19CH d1CK = 6 nM TAR1-5-19CK d1CH = 6 nMTAR1-5-19CH d2CK = 8 nM TAR1-5-19CH d3CK = 3 nM =12 nM TAR1-5 With(Gly₄Ser)_(n) linker heterodimers TAR1-5d1 = 30 nM TAR1-5d2 = 50 nMTAR1-5d3 = 300 nM TAR1-5d4 = 3 nM TAR1-5d5 = 200 nM TAR1-5d6 = 100 nM InFab format TAR1-5CH d2CK = 30 nM =60 nM TAR1-5CK d3CH = 100 nM TAR1-5-190.3 nM 3-10 nM (eg, 3 nM) homotrimer TAR2 TAR2 As TAR1 monomer As TAR1monomer 500 nM to 100 pM 500 nM to 50 pM monomers TAR2-10 TAR2-5 SerumAnti-SA 1 nM to 500 μM, 1 nM to 500 μM, Albumin monomers preferably 100nM to 10 μM preferably 100 nM to 10 μM In Dual Specific format, In DualSpecific format, target target affinity is 1 to affinity is 1 to 100,000x affinity 100,000 x affinity of SA of SA dAb affinity, eg 100 pM dAbaffinity, eg 100 pM (target) and 10 μM SA affinity. (target) and 10 μMSA affinity. MSA-16 200 nM MSA-26  70 nM

What we claim is:
 1. An isolated ligand comprising an antibody singlevariable domain comprising an amino acid sequence selected from thegroup consisting of: SEQ ID NO: 232 (dAb7h11) and an amino acid sequencethat is at least 95% identical to SEQ ID NO: 232, wherein at least oneof said antibody single variable domains is an antibody single variabledomain that specifically binds serum albumin.
 2. The isolated ligand ofclaim 1, wherein said at least one antibody single variable domain thatspecifically binds serum albumin binds to Domain II of serum albumin. 3.The isolated ligand of claim 2, wherein said ligand comprises a monomerantibody single variable domain which specifically binds to Domain II ofserum albumin.
 4. The isolated ligand of claim 3, wherein said monomerantibody single variable domain is conjugated to a drug.
 5. The isolatedligand of claim 2, wherein said ligand is a dual specific ligand,wherein said dual specific ligand comprises a first antibody singlevariable domain, wherein said first antibody single variable domainspecifically binds to Domain II of serum albumin.
 6. The isolated ligandof claim 5, wherein said dual specific ligand further comprises a secondantibody single variable domain, wherein said second antibody singlevariable domain specifically binds a target other than serum albumin. 7.An IgG comprising the dual-specific ligand of claim 6, wherein said IgGcomprises four antibody single variable domains.
 8. The isolated ligandof claim 1, wherein said antibody single variable domain comprises a setof four Kabat antibody framework regions (FRs), said set being encodedby human framework germ line antibody gene segments.
 9. An isolatedligand comprising at least one antibody single variable domain whichthat specifically binds serum albumin and also competes for binding toserum albumin with an antibody single variable domain which comprisesthe amino acid sequence of an antibody single variable domain of SEQ IDNO: 232 (dAb7h11).
 10. The isolated ligand of claim 6, wherein saidsecond antibody single variable domain is selected from the groupconsisting of: cytokines, cytokine receptors, enzymes, enzyme co-factorsand DNA binding proteins, wherein said second antibody single variabledomain specifically binds a target other than serum albumin.
 11. Theisolated ligand of claim 9, wherein said ligand is a dual specificligand, wherein said dual specific ligand comprises said antibody singlevariable domain that specifically binds serum albumin and a secondantibody single variable domain, wherein said second antibody singlevariable domain specifically binds a target other than serum albumin.12. The isolated ligand according to claim 1, wherein at least one ofsaid antibody single variable domains specifically binds an epitope orantigen with a dissociation constant (Kd) selected from the groupconsisting of: 1×10⁻³ M or less, 1×10⁻⁴M or less, 1×10⁻⁵ M or less,1×10⁻⁶M or less, 1×10⁻⁷M or less, 1×10⁻⁸ M or less, and 1×10⁻⁹ M orless, as determined by surface plasmon resonance.
 13. The isolatedligand of claim 1, wherein said ligand is a dual specific ligand,wherein said dual specific ligand comprises said antibody singlevariable domain that specifically binds serum albumin and a secondantibody single variable domain, wherein said second antibody singlevariable domain specifically binds a target other than serum albumin.14. The isolated ligand according to claim 1, wherein said ligandfurther comprises at least one entity selected from the group consistingof: a label, a tag and a drug.
 15. A composition comprising the isolatedligand according to claim 1, and a carrier thereof.
 16. The isolatedligand according to claim 6, wherein said ligand is a dual specificligand, and wherein: a. each of said antibody single variable domainsthat specifically binds serum albumin and said antibody single variabledomain that specifically binds a target other than serum albumin, is anantibody heavy chain single variable domain; or b. each of said antibodysingle variable domains that specifically binds serum albumin and saidantibody single variable domains that specifically binds a target otherthan serum albumin, is an antibody light chain single variable domain;or c. said antibody single variable domain that specifically binds serumalbumin is an antibody heavy chain single variable domain, and saidantibody single variable domain that specifically binds a target otherthan serum albumin is an antibody light chain single variable domain; ord. said antibody single variable domain that specifically binds serumalbumin is an antibody light chain single variable domain, and saidantibody single variable domain that specifically binds a target otherthan serum albumin is an antibody heavy chain single variable domain.17. The isolated ligand of any one of the claims according to claim 6,wherein said ligand is a dual specific ligand, and wherein: a. each ofsaid antibody single variable domains that specifically binds to DomainII of serum albumin and said antibody single variable domains thatspecifically binds a target other than serum albumin, is an antibodyheavy chain single variable domain; or b. each of said antibody singlevariable domain that specifically binds to Domain II of serum albuminand said antibody single variable domain that specifically binds atarget other than serum albumin, is an antibody light chain singlevariable domain; or c. said antibody single variable domain thatspecifically binds to Domain II of serum albumin is an antibody heavychain single variable domain, and said antibody single variable domainthat specifically binds an antigen other than serum albumin is anantibody light chain single variable domain; or d. said antibody singlevariable domain that specifically binds to Domain II of serum albumin isan antibody light chain single variable domain, and said antibody singlevariable domain that specifically binds an antigen other than serumalbumin is an antibody heavy chain single variable domain.
 18. Theisolated ligand of claim 9, wherein said at least one antibody singlevariable domain that specifically binds serum albumin binds to Domain IIof serum albumin.